Swine influenza A virus isolates containing the pandemic H1N1 origin matrix gene elicit greater disease in the murine model.

Since the 1990s, endemic North American swine influenza A viruses (swFLUAVs) contained an internal gene segment constellation, the triple reassortment internal gene (TRIG) cassette. In 2009, the H1N1 pandemic (pdmH1N1) virus spilled back into swine but did not become endemic. However, the pdmH1N1 contributed the matrix gene (pdmM) to the swFLUAVs circulating in the pig population, which replaced the classical swine matrix gene (swM) found in the TRIG cassette, suggesting the pdmM has a fitness benefit. Others have shown that swFLUAVs containing the pdmM have greater transmission efficiency compared to viruses containing the swM gene segment. We hypothesized that the matrix (M) gene could also affect disease and utilized two infection models, resistant BALB/c and susceptible DBA/2 mice, to assess pathogenicity. We infected BALB/c and DBA/2 mice with H1 and H3 swFLUAVs containing the swM or pdmM and measured lung virus titers, morbidity, mortality, and lung histopathology. H1 influenza strains containing the pdmM gene caused greater morbidity and mortality in resistant and susceptible murine strains, while H3 swFLUAVs caused no clinical disease. However, both H1 and H3 swFLUAVs containing the pdmM replicated to higher viral titers in the lungs and pdmM containing H1 viruses induced greater histological changes compared to swM H1 viruses. While the surface glycoproteins and other gene segments may contribute to swFLUAV pathogenicity in mice, these data suggest that the origin of the matrix gene also contributes to pathogenicity of swFLUAV in mice, although we must be cautious in translating these conclusions to their natural host, swine. IMPORTANCE: The 2009 pandemic H1N1 virus rapidly spilled back into North American swine, reassorting with the already genetically diverse swFLUAVs. Notably, the M gene segment quickly replaced the classical M gene segment, suggesting a fitness benefit. Here, using two murine models of infection, we demonstrate that swFLUAV isolates containing the pandemic H1N1 origin M gene caused increased disease compared to isolates containing the classical swine M gene. These results suggest that, in addition to other influenza virus gene segments, the swFLUAV M gene segment contributes to pathogenesis in mammals.

Viral expansion after transfer is a primary driver of influenza A virus transmission bottlenecks.

For many viruses, narrow bottlenecks acting during transmission sharply reduce genetic diversity in a recipient host relative to the donor. Since genetic diversity represents adaptive potential, such losses of diversity are though to limit the opportunity for viral populations to undergo antigenic change and other adaptive processes. Thus, a detailed picture of evolutionary dynamics during transmission is critical to understanding the forces driving viral evolution at an epidemiologic scale. To advance this understanding, we used a novel barcoded virus library and a guinea pig model of transmission to decipher where in the transmission process diversity is lost for influenza A viruses. In inoculated guinea pigs, we show that a high level of viral genetic diversity is maintained across time. Continuity in the barcodes detected furthermore indicates that stochastic effects are not pronounced within inoculated hosts. Importantly, in both aerosol-exposed and direct contact-exposed animals, we observed many barcodes at the earliest time point(s) positive for infectious virus, indicating robust transfer of diversity through the environment. This high viral diversity is short-lived, however, with a sharp decline seen 1-2 days after initiation of infection. Although major losses of diversity at transmission are well described for influenza A virus, our data indicate that events that occur following viral transfer and during the earliest stages of natural infection have a predominant role in this process. This finding suggests that immune selection may have greater opportunity to operate during influenza A transmission than previously recognized.

Antigenic mapping of the hemagglutinin of the H9 subtype influenza A viruses using sera from Japanese quail (Coturnix c. japonica).

IMPORTANCE: Determining the relevant amino acids involved in antigenic drift on the surface protein hemagglutinin (HA) is critical to understand influenza virus evolution and efficient assessment of vaccine strains relative to current circulating strains. We used antigenic cartography to generate an antigenic map of the H9 hemagglutinin (HA) using sera produced in one of the most relevant minor poultry species, Japanese quail. Key antigenic positions were identified and tested to confirm their impact on the antigenic profile. This work provides a better understanding of the antigenic diversity of the H9 HA as it relates to reactivity to quail sera and will facilitate a rational approach for selecting more efficacious vaccines against poultry-origin H9 influenza viruses in minor poultry species.

South American H4N2 influenza A virus improved replication in chicken trachea after low number of passages.

Introduction of influenza A viruses (FLUAV) into poultry from waterfowl is frequent, producing economic burden and increasing the probability of human infections. We have previously described the presence of FLUAV in wild birds in Argentina with unique evolutionary trajectories belonging to a South American lineage different from the North American and Eurasian lineages. Adaptability of this South American lineage FLUAV to poultry species is still poorly understood. In the present report, we evaluated the capacity of an H4N2 FLUAV from the South American lineage to adapt to chickens after low number of passages. We found that five mutations were acquired after five passages in 3-days-old chickens. These mutations produced a virus with better infectivity in ex vivo trachea explants but overall lower infection in lung explants. Infection of 3-week-old chickens persisted for a longer period and was detected in more tissues than the parental virus, suggesting adaptation of the H4N2 influenza A virus to chicken.

Epistasis reduces fitness costs of influenza A virus escape from stem-binding antibodies.

The hemagglutinin (HA) stem region is a major target of universal influenza vaccine efforts owing to the presence of highly conserved epitopes across multiple influenza A virus (IAV) strains and subtypes. To explore the potential impact of vaccine-induced immunity targeting the HA stem, we examined the fitness effects of viral escape from stem-binding broadly neutralizing antibodies (stem-bnAbs). Recombinant viruses containing each individual antibody escape substitution showed diminished replication compared to wild-type virus, indicating that stem-bnAb escape incurred fitness costs. A second-site mutation in the HA head domain (N129D; H1 numbering) reduced the fitness effects observed in primary cell cultures and likely enabled the selection of escape mutations. Functionally, this putative permissive mutation increased HA avidity for its receptor. These results suggest a mechanism of epistasis in IAV, wherein modulating the efficiency of attachment eases evolutionary constraints imposed by the requirement for membrane fusion. Taken together, the data indicate that viral escape from stem-bnAbs is costly but highlights the potential for epistatic interactions to enable evolution within the functionally constrained HA stem domain.

Exclusion of latecomers yields a patchwork of viral subpopulations within hosts.

Viruses arriving late to an individual cell are blocked from replicating, an effect called superinfection exclusion. A study in PLOS Biology indicates that this exclusion at the level of individual cells gives rise to a heterogenous landscape of infection within a host.

Transmission of Human Influenza A Virus in Pigs Selects for Adaptive Mutations on the HA Gene.

Influenza A viruses (FLUAV) cause respiratory diseases in many host species, including humans and pigs. The spillover of FLUAV between swine and humans has been a concern for both public health and the swine industry. With the emergence of the triple reassortant internal gene (TRIG) constellation, establishment of human-origin FLUAVs in pigs has become more common, leading to increased viral diversity. However, little is known about the adaptation processes that are needed for a human-origin FLUAV to transmit and become established in pigs. We generated a reassortant FLUAV (VIC11pTRIG) containing surface gene segments from a human FLUAV strain and internal gene segments from the 2009 pandemic and TRIG FLUAV lineages and demonstrated that it can replicate and transmit in pigs. Sequencing and variant analysis identified three mutants that emerged during replication in pigs, which were mapped near the receptor binding site of the hemagglutinin (HA). The variants replicated more efficiently in differentiated swine tracheal cells compared to the virus containing the wildtype human-origin HA, and one of them was present in all contact pigs. These results show that variants are selected quickly after replication of human-origin HA in pigs, leading to improved fitness in the swine host, likely contributing to transmission. IMPORTANCE Influenza A viruses cause respiratory disease in several species, including humans and pigs. The bidirectional transmission of FLUAV between humans and pigs plays a significant role in the generation of novel viral strains, greatly impacting viral epidemiology. However, little is known about the evolutionary processes that allow human FLUAV to become established in pigs. In this study, we generated reassortant viruses containing human seasonal HA and neuraminidase (NA) on different constellations of internal genes and tested their ability to replicate and transmit in pigs. We demonstrated that a virus containing a common internal gene constellation currently found in U.S. swine was able to transmit efficiently via the respiratory route. We identified a specific amino acid substitution that was fixed in the respiratory contact pigs that was associated with improved replication in primary swine tracheal epithelial cells, suggesting it was crucial for the transmissibility of the human virus in pigs.

Influenza A virus reassortment in mammals gives rise to genetically distinct within-host subpopulations.

Influenza A virus (IAV) genetic exchange through reassortment has the potential to accelerate viral evolution and has played a critical role in the generation of multiple pandemic strains. For reassortment to occur, distinct viruses must co-infect the same cell. The spatio-temporal dynamics of viral dissemination within an infected host therefore define opportunity for reassortment. Here, we used wild type and synonymously barcoded variant viruses of a pandemic H1N1 strain to examine the within-host viral dynamics that govern reassortment in guinea pigs, ferrets and swine. The first two species are well-established models of human influenza, while swine are a natural host and a frequent conduit for cross-species transmission and reassortment. Our results show reassortment to be pervasive in all three hosts but less frequent in swine than in ferrets and guinea pigs. In ferrets, tissue-specific differences in the opportunity for reassortment are also evident, with more reassortants detected in the nasal tract than the lower respiratory tract. While temporal trends in viral diversity are limited, spatial patterns are clear, with heterogeneity in the viral genotypes detected at distinct anatomical sites revealing extensive compartmentalization of reassortment and replication. Our data indicate that the dynamics of viral replication in mammals allow diversification through reassortment but that the spatial compartmentalization of variants likely shapes their evolution and onward transmission.

Timing of exposure is critical in a highly sensitive model of SARS-CoV-2 transmission.

Transmission efficiency is a critical factor determining the size of an outbreak of infectious disease. Indeed, the propensity of SARS-CoV-2 to transmit among humans precipitated and continues to sustain the COVID-19 pandemic. Nevertheless, the number of new cases among contacts is highly variable and underlying reasons for wide-ranging transmission outcomes remain unclear. Here, we evaluated viral spread in golden Syrian hamsters to define the impact of temporal and environmental conditions on the efficiency of SARS-CoV-2 transmission through the air. Our data show that exposure periods as brief as one hour are sufficient to support robust transmission. However, the timing after infection is critical for transmission success, with the highest frequency of transmission to contacts occurring at times of peak viral load in the donor animals. Relative humidity and temperature had no detectable impact on transmission when exposures were carried out with optimal timing and high inoculation dose. However, contrary to expectation, trends observed with sub-optimal exposure timing and lower inoculation dose suggest improved transmission at high relative humidity or high temperature. In sum, among the conditions tested, our data reveal the timing of exposure to be the strongest determinant of SARS-CoV-2 transmission success and implicate viral load as an important driver of transmission.

Intra- and inter-host evolution of H9N2 influenza A virus in Japanese quail.

Influenza A viruses (IAVs) are constantly evolving. Crucial steps in the infection cycle, such as sialic acid (SA) receptor binding on the host cell surface, can either promote or hamper the emergence of new variants. We previously assessed the relative fitness in Japanese quail of H9N2 variant viruses differing at a single amino acid position, residue 216 in the hemagglutinin (HA) viral surface protein. This site is known to modulate SA recognition. Our prior study generated a valuable set of longitudinal samples from quail transmission groups where the inoculum comprised different mixed populations of HA 216 variant viruses. Here, we leveraged these samples to examine the evolutionary dynamics of viral populations within and between inoculated and naïve contact quails. We found that positive selection dominated HA gene evolution, but fixation of the fittest variant depended on the competition mixture. Analysis of the whole genome revealed further evidence of positive selection acting both within and between hosts. Positive selection drove fixation of variants in non-HA segments within inoculated and contact quails. Importantly, transmission bottlenecks were modulated by the molecular signature at HA 216, revealing viral receptor usage as a determinant of transmitted diversity. Overall, we show that selection strongly shaped the evolutionary dynamics within and between quails. These findings support the notion that selective processes act effectively on IAV populations in poultry hosts, facilitating rapid viral evolution in this ecological niche.

Mutation E48K in PB1 Polymerase Subunit Improves Stability of a Candidate Live Attenuated Influenza B Virus Vaccine.

Influenza B virus (IBV) is a major respiratory pathogen of humans, particularly in the elderly and children, and vaccines are the most effective way to control it. In previous work, incorporation of two mutations (E580G, S660A) along with the addition of an HA epitope tag in the PB1 segment of B/Brisbane/60/2008 (B/Bris) resulted in an attenuated strain that was safe and effective as a live attenuated vaccine. A third attempted mutation (K391E) in PB1 was not always stable. Interestingly, viruses that maintained the K391E mutation were associated with the mutation E48K. To explore the contribution of the E48K mutation to stability of the K391E mutation, a vaccine candidate was generated by inserting both mutations, along with attenuating mutations E580G and S660A, in PB1 of B/Bris (B/Bris PB1att 4M). Serial passages of the B/Bris PB1att 4M vaccine candidate in eggs and MDCK indicated high stability. In silico structural analysis revealed a potential interaction between amino acids at positions 48 and 391. In mice, B/Bris PB1att 4M was safe and provided complete protection against homologous challenge. These results confirm the compensatory effect of mutation E48K to stabilize the K391E mutation, resulting in a safer, yet still protective, IBV LAIV vaccine.

Development of a Novel Live Attenuated Influenza A Virus Vaccine Encoding the IgA-Inducing Protein.

Live attenuated influenza virus (LAIV) vaccines elicit a combination of systemic and mucosal immunity by mimicking a natural infection. To further enhance protective mucosal responses, we incorporated the gene encoding the IgA-inducing protein (IGIP) into the LAIV genomes of the cold-adapted A/Leningrad/134/17/57 (H2N2) strain (caLen) and the experimental attenuated backbone A/turkey/Ohio/313053/04 (H3N2) (OH/04att). Incorporation of IGIP into the caLen background led to a virus that grew poorly in prototypical substrates. In contrast, IGIP in the OH/04att background (IGIP-H1att) virus grew to titers comparable to the isogenic backbone H1att (H1N1) without IGIP. IGIP-H1att- and H1caLen-vaccinated mice were protected against lethal challenge with a homologous virus. The IGIP-H1att vaccine generated robust serum HAI responses in naïve mice against the homologous virus, equal or better than those obtained with the H1caLen vaccine. Analyses of IgG and IgA responses using a protein microarray revealed qualitative differences in humoral and mucosal responses between vaccine groups. Overall, serum and bronchoalveolar lavage samples from the IGIP-H1att group showed trends towards increased stimulation of IgG and IgA responses compared to H1caLen samples. In summary, the introduction of genes encoding immunomodulatory functions into a candidate LAIV can serve as natural adjuvants to improve overall vaccine safety and efficacy.

Collective interactions augment influenza A virus replication in a host-dependent manner.

Infection with a single influenza A virus (IAV) is only rarely sufficient to initiate productive infection. Instead, multiple viral genomes are often required in a given cell. Here, we show that the reliance of IAV on multiple infection can form an important species barrier. Namely, we find that avian H9N2 viruses representative of those circulating widely at the poultry-human interface exhibit acute dependence on collective interactions in mammalian systems. This need for multiple infection is greatly reduced in the natural host. Quantification of incomplete viral genomes showed that their complementation accounts for the moderate reliance on multiple infection seen in avian cells but not the added reliance seen in mammalian cells. An additional form of virus-virus interaction is needed in mammals. We find that the PA gene segment is a major driver of this phenotype and that both viral replication and transcription are affected. These data indicate that multiple distinct mechanisms underlie the reliance of IAV on multiple infection and underscore the importance of virus-virus interactions in IAV infection, evolution and emergence.

Silent Infection of B and CD8+ T Lymphocytes by Influenza A Virus in Children with Tonsillar Hypertrophy.

Influenza A viruses (IAVs) cause more than 2 million annual episodes of seasonal acute respiratory infections (ARI) and approximately 500,000 deaths worldwide. Depending on virus strain and host immune status, acute infections by IAV may reach sites other than the respiratory tract. In the present study, IAV RNA and antigens were searched for in tissues of palatine tonsils and adenoids removed from patients without ARI symptoms. A real-time reverse transcriptase PCR (RT-PCR) screening revealed that 8 tissue samples from 7 patients out of 103 were positive for IAV. Positive samples were subjected to next-generation sequencing (NGS) and 3 of 8 tissues yielded complete IAV pH1N1 genomes, whereas in 5 samples, the PB1 gene was not fully assembled. Phylogenetic analysis placed tonsil-derived IAV in clusters clearly segregated from contemporaneous Brazilian viruses. Flow cytometry of dispersed tissue fragments and serial immunohistochemistry of paraffin-embedded sections of naturally infected biopsies indicated that CD20+ B lymphocytes, CD8+ T lymphocytes, and CD11c+ cells are susceptible to IAV infection. We sought to investigate whether these lymphoid tissues could be sites of viral replication and sources of viable virus particles. MDCK cells were inoculated with tissue lysates, enabling recovery of one IAV isolate confirmed by immunofluorescence, reverse transcriptase quantitative PCR (RT-qPCR), and NGS. The data indicate that lymphoid tissues not only harbor expression of IAV proteins but also contain infectious virus. Asymptomatic long-term infection raises the possibility of IAV shedding from tonsils, which may have an impact on host-to-host transmission.IMPORTANCE Influenza A virus (IAV) infections are important threats to human health worldwide. Although extensively studied, some aspects of virus pathogenesis and tissue tropism remain unclear. Here, by different strategies, we describe the asymptomatic infection of human lymphoid organs by IAV in children. Our results indicate that IAV was not only detected and isolated from human tonsils but displayed unique genetic features in comparison with those of contemporaneous IAVs circulating in Brazil and detected in swabs and nasal washes. Inside the tissue microenvironment, immune cells were shown to be carrying IAV antigens, especially B and T CD8+ lymphocytes. Taken together, these results suggest that human lymphoid tissues can be sites of silent IAV infections with possible impact on virus shedding to the population.

Improved detection of influenza A virus from blue-winged teals by sequencing directly from swab material.

ABSTRACT: The greatest diversity of influenza A virus (IAV) is found in wild aquatic birds of the orders Anseriformes and Charadriiformes. In these birds, IAV replication occurs mostly in the intestinal tract. Fecal, cloacal, and/or tracheal swabs are typically collected and tested by real-time RT-PCR (rRT-PCR) and/or by virus isolation in embryonated chicken eggs in order to determine the presence of IAV. Virus isolation may impose bottlenecks that select variant populations that are different from those circulating in nature, and such bottlenecks may result in artifactual representation of subtype diversity and/or underrepresented mixed infections. The advent of next-generation sequencing (NGS) technologies provides an opportunity to explore to what extent IAV subtype diversity is affected by virus isolation in eggs. In the present work, we evaluated the advantage of sequencing by NGS directly from swab material of IAV rRT-PCR-positive swabs collected during the 2013-14 surveillance season in Guatemala and compared to results from NGS after virus isolation. The results highlight the benefit of sequencing IAV genomes directly from swabs to better understand subtype diversity and detection of alternative amino acid motifs that could otherwise escape detection using traditional methods of virus isolation. In addition, NGS sequencing data from swabs revealed reduced presence of defective interfering particles compared to virus isolates. We propose an alternative workflow in which original swab samples positive for IAV by rRT-PCR are first subjected to NGS before attempting viral isolation. This approach should speed the processing of samples and better capture natural IAV diversity. OPEN RESEARCH BADGES: This article has earned an Open Data Badge for making publicly available the digitally-shareable data necessary to reproduce the reported results. The data is available at https://doi.org/10.5061/dryad.3h2n106.

Expression and strain variation of the novel "small open reading frame" (smorf) multigene family in Babesia bovis.

Small open reading frame (smorf) genes comprise the second largest Babesia bovis multigene family. All known 44 variant smorf genes are located in close chromosomal proximity to ves1 genes, which encode proteins that mediate cytoadhesion and contribute to immune evasion. In this study, we characterised the general topology of smorf genes and investigated the gene repertoire, transcriptional profile and SMORF expression in two distinct strains, T2Bo and Mo7. Sequence analysis using degenerate primers identified additional smorf genes in each strain and demonstrated that the smorf gene repertoire varies between strains, with conserved and unique genes in both. Smorf genes have multiple semi-conserved and variable blocks, and a large hypervariable insertion in 20 of the 44 genes defines two major branches of the family, termed smorf A and smorf B. A total of 32 smorf genes are simultaneously transcribed in T2Bo strain B. bovis merozoites obtained from deep brain tissue of an acutely infected animal. SMORF peptide-specific antiserum bound in immunoblots to multiple proteins with a range of sizes predicted by smorf genes, confirming translation of smorf gene products from these transcripts. These results indicate that the smorf multigene family is larger than previously described and demonstrate that smorf genes are expressed and are undergoing variation, both within strains and in a lineage-specific pattern independent of strain specificity. The function of these novel proteins is unknown.