Nucleoprotein reassortment enhanced transmissibility of H3 1990.4.a clade influenza A virus in swine.

The increased detection of H3 C-IVA (1990.4.a) clade influenza A viruses (IAVs) in US swine in 2019 was associated with a reassortment event to acquire an H1N1pdm09 lineage nucleoprotein (pdmNP) gene, replacing a TRIG lineage NP (trigNP). We hypothesized that acquiring the pdmNP conferred a selective advantage over prior circulating H3 viruses with a trigNP. To investigate the role of NP reassortment in transmission, we identified two contemporary 1990.4.a representative strains (NC/19 and MN/18) with different evolutionary origins of the NP gene. A reverse genetics system was used to generate wild-type (wt) strains and swap the pdm and TRIG lineage NP genes, generating four viruses: wtNC/19-pdmNP, NC/19-trigNP, wtMN/18-trigNP, and MN/18-pdmNP. The pathogenicity and transmission of the four viruses were compared in pigs. All four viruses infected 10 primary pigs and transmitted to five indirect contact pigs per group. Pigs infected via contact with MN/18-pdmNP shed virus 2 days earlier than pigs infected with wtMN/18-trigNP. The inverse did not occur for wtNC/19-pdmNP and NC/19-trigNP. This suggests that pdmNP reassortment resulted in a combination of genes that improved transmission efficiency when paired with the 1990.4.a hemagglutinin (HA). This is likely a multigenic trait, as replacing the trigNP gene did not diminish the transmission of a wild-type IAV in swine. This study demonstrates how reassortment and evolutionary change of internal genes can result in more transmissible viruses that influence HA clade detection frequency. Thus, rapidly identifying novel reassortants paired with dominant hemagglutinin/neuraminidase may improve the prediction of strains to include in vaccines.IMPORTANCEInfluenza A viruses (IAVs) are composed of eight non-continuous gene segments that can reassort during coinfection of a host, creating new combinations. Some gene combinations may convey a selective advantage and be paired together preferentially. A reassortment event was detected in swine in the United States that involved the exchange of two lineages of nucleoprotein (NP) genes (trigNP to pdmNP) that became a predominant genotype detected in surveillance. Using a transmission study, we demonstrated that exchanging the trigNP for a pdmNP caused the virus to shed from the nose at higher levels and transmit to other pigs more rapidly. Replacing a pdmNP with a trigNP did not hinder transmission, suggesting that transmission efficiency depends on interactions between multiple genes. This demonstrates how reassortment alters IAV transmission and that reassortment events can provide an explanation for why genetically related viruses with different internal gene combinations experience rapid fluxes in detection frequency.

Impact of sex on humoral immunity with live influenza B virus vaccines in mice.

Influenza B virus (FLUBV) poses a significant infectious threat, with frequent vaccine mismatch limiting its effectiveness. Our previous work investigated the safety and efficacy of modified live attenuated FLUBV vaccines with rearranged genomes (FluB-RAM and FluB-RANS) or a temperature-sensitive PB1 segment with a C-terminal HA tag (FluB-att). In this study, we compared the immune responses of female and male DBA/2J mice vaccinated with these vaccines, including versions containing a chimeric HA segment with an N-terminal IgA-inducing peptide (IGIP). Importantly, both recombinant viruses with and without IGIP remained genetically stable during egg passage. We found that introducing IGIP strengthened vaccine attenuation, particularly for FluB-RAM/IGIP. Prime-boost vaccination completely protected mice against lethal challenge with a homologous FLUBV strain. Notably, recombinant viruses induced robust neutralizing antibody responses (hemagglutination inhibition titers ≥40) alongside antibodies against NA and NP. Interestingly, female mice displayed a consistent trend of enhanced humoral and cross-reactive IgG and IgA responses against HA, NA, and NP compared to male counterparts, regardless of the vaccine used. However, the presence of IGIP generally led to lower anti-HA responses but higher anti-NA and anti-NP responses, particularly of the IgA isotype. These trends were further reflected in mucosal and serological responses two weeks after challenge, with clear distinctions based on sex, vaccine backbone, and IGIP inclusion. These findings hold significant promise for advancing the development of universal influenza vaccines.

Amino acid 138 in the HA of a H3N2 subtype influenza A virus increases affinity for the lower respiratory tract and alveolar macrophages in pigs.

Influenza A virus (FLUAV) infects a wide range of hosts and human-to-swine spillover events are frequently reported. However, only a few of these human viruses have become established in pigs and the host barriers and molecular mechanisms driving adaptation to the swine host remain poorly understood. We previously found that infection of pigs with a 2:6 reassortant virus (hVIC/11) containing the hemagglutinin (HA) and neuraminidase (NA) gene segments from the human strain A/Victoria/361/2011 (H3N2) and internal gene segments of an endemic swine strain (sOH/04) resulted in a fixed amino acid substitution in the HA (A138S, mature H3 HA numbering). In silico analysis revealed that S138 became predominant among swine H3N2 virus sequences deposited in public databases, while 138A predominates in human isolates. To understand the role of the HA A138S substitution in the adaptation of a human-origin FLUAV HA to swine, we infected pigs with the hVIC/11A138S mutant and analyzed pathogenesis and transmission compared to hVIC/11 and sOH/04. Our results showed that the hVIC/11A138S virus had an intermediary pathogenesis between hVIC/11 and sOH/04. The hVIC/11A138S infected the upper respiratory tract, right caudal, and both cranial lobes while hVIC/11 was only detected in nose and trachea samples. Viruses induced a distinct expression pattern of various pro-inflammatory cytokines such as IL-8, TNF-α, and IFN-ß. Flow cytometric analysis of lung samples revealed a significant reduction of porcine alveolar macrophages (PAMs) in hVIC/11A138S-infected pigs compared to hVIC/11 while a MHCIIlowCD163neg population was increased. The hVIC/11A138S showed a higher affinity for PAMs than hVIC/11, noted as an increase of infected PAMs in bronchoalveolar lavage fluid (BALF), and showed no differences in the percentage of HA-positive PAMs compared to sOH/04. This increased infection of PAMs led to an increase of granulocyte-monocyte colony-stimulating factor (GM-CSF) stimulation but a reduced expression of peroxisome proliferator-activated receptor gamma (PPARγ) in the sOH/04-infected group. Analysis using the PAM cell line 3D4/21 revealed that the A138S substitution improved replication and apoptosis induction in this cell type compared to hVIC/11 but at lower levels than sOH/04. Overall, our study indicates that adaptation of human viruses to the swine host involves an increased affinity for the lower respiratory tract and alveolar macrophages.

FLUAV RAM-IGIP: A modified live influenza virus vaccine that enhances humoral and mucosal responses against influenza.

Current influenza A vaccines fall short, leaving both humans and animals vulnerable. To address this issue, we have developed attenuated modified live virus (MLV) vaccines against influenza using genome rearrangement techniques targeting the internal gene segments of FLUAV. The rearranged M2 (RAM) strategy involves cloning the M2 ORF downstream of the PB1 ORF in segment 2 and incorporating multiple early stop codons within the M2 ORF in segment 7. Additionally, the IgA-inducing protein (IGIP) coding region was inserted into the HA segment to further attenuate the virus and enhance protective mucosal responses. RAM-IGIP viruses exhibit similar growth rates to wild type (WT) viruses in vitro and remain stable during multiple passages in cells and embryonated eggs. The safety, immunogenicity, and protective efficacy of the RAM-IGIP MLV vaccine against the prototypical 2009 pandemic H1N1 strain A/California/04/2009 (H1N1) (Ca/04) were evaluated in Balb/c mice and compared to a prototypic cold-adapted live attenuated virus vaccine. The results demonstrate that the RAM-IGIP virus exhibits attenuated virulence in vivo. Mice vaccinated with RAM-IGIP and subsequently challenged with an aggressive lethal dose of the Ca/04 strain exhibited complete protection. Analysis of the humoral immune response revealed that the inclusion of IGIP enhanced the production of neutralizing antibodies and augmented the antibody-dependent cellular cytotoxicity response. Similarly, the RAM-IGIP potentiated the mucosal immune response against various FLUAV subtypes. Moreover, increased antibodies against NP and NA responses were observed. These findings support the development of MLVs utilizing genome rearrangement strategies in conjunction with the incorporation of immunomodulators. IMPORTANCE: Current influenza vaccines offer suboptimal protection, leaving both humans and animals vulnerable. Our novel attenuated MLV vaccine, built by rearranging FLUAV genome segments and incorporating the IgA-inducing protein, shows promising results. This RAM-IGIP vaccine exhibits safe attenuation, robust immune responses, and complete protection against lethal viral challenge in mice. Its ability to stimulate broad-spectrum humoral and mucosal immunity against diverse FLUAV subtypes makes it a highly promising candidate for improved influenza vaccines.

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.

Characterizing a century of genetic diversity and contemporary antigenic diversity of N1 neuraminidase in influenza A virus from North American swine.

Influenza A viruses (IAVs) of the H1N1 classical swine lineage became endemic in North American swine following the 1918 pandemic. Additional human-to-swine transmission events after 1918, and a spillover of H1 viruses from wild birds in Europe, potentiated a rapid increase in genomic diversity via reassortment between introductions and the endemic classical swine lineage. To determine mechanisms affecting reassortment and evolution, we conducted a phylogenetic analysis of N1 and paired HA swine IAV genes in North America between 1930 and 2020. We described fourteen N1 clades within the N1 Eurasian avian lineage (including the N1 pandemic clade), the N1 classical swine lineage, and the N1 human seasonal lineage. Seven N1 genetic clades had evidence for contemporary circulation. To assess antigenic drift associated with N1 genetic diversity, we generated a panel of representative swine N1 antisera and quantified the antigenic distance between wild-type viruses using enzyme-linked lectin assays and antigenic cartography. Within the N1 genes, the antigenic similarity was variable and reflected shared evolutionary history. Sustained circulation and evolution of N1 genes in swine had resulted in a significant antigenic distance between the N1 pandemic clade and the classical swine lineage. Between 2010 and 2020, N1 clades and N1-HA pairings fluctuated in detection frequency across North America, with hotspots of diversity generally appearing and disappearing within 2 years. We also identified frequent N1-HA reassortment events (n = 36), which were rarely sustained (n = 6) and sometimes also concomitant with the emergence of new N1 genetic clades (n = 3). These data form a baseline from which we can identify N1 clades that expand in range or genetic diversity that may impact viral phenotypes or vaccine immunity and subsequently the health of North American swine.

Blue-Winged Teals in Guatemala and Their Potential Role in the Ecology of H14 Subtype Influenza a Viruses.

Wild aquatic birds are considered the natural hosts of 16 HA (H1-H16) and 9 NA (N1-N9) subtypes of influenza A viruses (FLUAV) found in different combinations. H14 FLUAVs are rarely detected in nature. Since 2011, H14 FLUAVs have been consistently detected in Guatemala, leading to the largest collection of this subtype from a single country. All H14 FLUAVs in Guatemala were detected from blue-winged teal samples. In this report, 17 new full-length H14 FLUAV genome sequences detected from 2014 until 2019 were analyzed and compared to all published H14 sequences, including Guatemala, North America, and Eurasia. The H14 FLUAVs identified in Guatemala were mostly associated with the N3 subtype (n = 25), whereas the rest were paired with either N4 (n = 7), N5 (n = 4), N6 (n = 1), and two mixed infections (N3/N5 n = 2, and N2/N3 n = 1). H14 FLUAVs in Guatemala belong to a distinct H14 lineage in the Americas that is evolving independently from the Eurasian H14 lineage. Of note, the ORF of the H14 HA segments showed three distinct motifs at the cleavage site, two of these containing arginine instead of lysine in the first and fourth positions, not previously described in other countries. The effects of these mutations on virus replication, virulence, and/or transmission remain unknown and warrant further studies.

Evolution and Introductions of Influenza A Virus H1N1 in a Farrow-to-Finish Farm in Guatemala.

Commercial swine farms provide unique systems for interspecies transmission of influenza A viruses (FLUAVs) at the animal-human interface. Bidirectional transmission of FLUAVs between pigs and humans plays a significant role in the generation of novel strains that become established in the new host population. Active FLUAV surveillance was conducted for 2 years on a commercial pig farm in Southern Guatemala with no history of FLUAV vaccination. Nasal swabs (n = 2,094) from fattening pigs (6 to 24 weeks old) with respiratory signs were collected weekly from May 2016 to February 2018. Swabs were screened for FLUAV by real-time reverse transcriptase PCR (RRT-PCR), and full virus genomes of FLUAV-positive swabs were sequenced by next-generation sequencing (NGS). FLUAV prevalence was 12.0% (95% confidence interval [CI], 10.6% to 13.4%) with two distinct periods of high infection. All samples were identified as FLUAVs of the H1N1 subtype within the H1 swine clade 1A.3.3.2 and whose ancestors are the human origin 2009 H1N1 influenza pandemic virus (H1N1 pdm09). Compared to the prototypic reference segment sequence, 10 amino acid signatures were observed on relevant antigenic sites on the hemagglutinin. The Guatemalan swine-origin FLUAVs show independent evolution from other H1N1 pdm09 FLUAVs circulating in Central America. The zoonotic risk of these viruses remains unknown but strongly calls for continued FLUAV surveillance in pigs in Guatemala. IMPORTANCE Despite increased surveillance efforts, the epidemiology of FLUAVs circulating in swine in Latin America remains understudied. For instance, the 2009 H1N1 influenza pandemic strain (H1N1 pdm09) emerged in Mexico, but its circulation remained undetected in pigs. In Central America, Guatemala is the country with the largest swine industry. We found a unique group of H1N1 pdm09 sequences that suggests independent evolution from similar viruses circulating in Central America. These viruses may represent the establishment of a novel genetic lineage with the potential to reassort with other cocirculating viruses and whose zoonotic risk remains to be determined.

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.

Pathobiology and dysbiosis of the respiratory and intestinal microbiota in 14 months old Golden Syrian hamsters infected with SARS-CoV-2.

The pandemic of severe acute respiratory syndrome coronavirus 2 (SARS2) affected the geriatric population. Among research models, Golden Syrian hamsters (GSH) are one of the most representative to study SARS2 pathogenesis and host responses. However, animal studies that recapitulate the effects of SARS2 in the human geriatric population are lacking. To address this gap, we inoculated 14 months old GSH with a prototypic ancestral strain of SARS2 and studied the effects on virus pathogenesis, virus shedding, and respiratory and gastrointestinal microbiome changes. SARS2 infection led to high vRNA loads in the nasal turbinates (NT), lungs, and trachea as well as higher pulmonary lesions scores later in infection. Dysbiosis throughout SARS2 disease progression was observed in the pulmonary microbial dynamics with the enrichment of opportunistic pathogens (Haemophilus, Fusobacterium, Streptococcus, Campylobacter, and Johnsonella) and microbes associated with inflammation (Prevotella). Changes in the gut microbial community also reflected an increase in multiple genera previously associated with intestinal inflammation and disease (Helicobacter, Mucispirillum, Streptococcus, unclassified Erysipelotrichaceae, and Spirochaetaceae). Influenza A virus (FLUAV) pre-exposure resulted in slightly more pronounced pathology in the NT and lungs early on (3 dpc), and more notable changes in lungs compared to the gut microbiome dynamics. Similarities among aged GSH and the microbiome in critically ill COVID-19 patients, particularly in the lower respiratory tract, suggest that GSHs are a representative model to investigate microbial changes during SARS2 infection. The relationship between the residential microbiome and other confounding factors, such as SARS2 infection, in a widely used animal model, contributes to a better understanding of the complexities associated with the host responses during viral infections.

Changes in the Hemagglutinin and Internal Gene Segments Were Needed for Human Seasonal H3 Influenza A Virus to Efficiently Infect and Replicate in Swine.

The current diversity of influenza A viruses (IAV) circulating in swine is largely a consequence of human-to-swine transmission events and consequent evolution in pigs. However, little is known about the requirements for human IAVs to transmit to and subsequently adapt in pigs. Novel human-like H3 viruses were detected in swine herds in the U.S. in 2012 and have continued to circulate and evolve in swine. We evaluated the contributions of gene segments on the ability of these viruses to infect pigs by using a series of in vitro models. For this purpose, reassortant viruses were generated by reverse genetics (rg) swapping the surface genes (hemagglutinin-HA and neuraminidase-NA) and internal gene segment backbones between a human-like H3N1 isolated from swine and a seasonal human H3N2 virus with common HA ancestry. Virus growth kinetics in porcine intestinal epithelial cells (SD-PJEC) and in ex-vivo porcine trachea explants were significantly reduced by replacing the swine-adapted HA with the human seasonal HA. Unlike the human HA, the swine-adapted HA demonstrated more abundant attachment to epithelial cells throughout the swine respiratory tract by virus histochemistry and increased entry into SD-PJEC swine cells. The human seasonal internal gene segments improved replication of the swine-adapted HA at 33 °C, but decreased replication at 40 °C. Although the HA was crucial for the infectivity in pigs and swine tissues, these results suggest that the adaptation of human seasonal H3 viruses to swine is multigenic and that the swine-adapted HA alone was not sufficient to confer the full phenotype of the wild-type swine-adapted virus.

Phylogenetic analysis and comparative genomics of SARS-CoV-2 from survivor and non-survivor COVID-19 patients in Cordoba, Argentina.

BACKGROUND: The SARS-CoV-2 virus is responsible for the COVID-19 pandemic. To better understand the evolution of SARS-CoV-2 early in the pandemic in the Province of Cordoba, Argentina, we performed a comparative genomic analysis of SARS-CoV-2 strains detected in survivors and non-survivors of COVID-19. We also carried out an epidemiological study to find a possible association between the symptoms and comorbidities of these patients with their clinical outcomes. RESULTS: A representative sampling was performed in different cities in the Province of Cordoba. Ten and nine complete SARS-CoV-2 genomes were obtained by next-generation sequencing of nasopharyngeal specimens from non-survivors and survivors, respectively. Phylogenetic and phylodynamic analyses revealed multiple introductions of the most common lineages in South America, including B.1, B.1.1.1, B.1.499, and N.3. Fifty-six mutations were identified, with 14% of those in common between the non-survivor and survivor groups. Specific SARS-CoV-2 mutations for survivors constituted 25% whereas for non-survivors they were 41% of the repertoire, indicating partial selectivity. The non-survivors' variants showed higher diversity in 9 genes, with a majority in Nsp3, while the survivors' variants were detected in 5 genes, with a higher incidence in the Spike protein. At least one comorbidity was present in 60% of non-survivor patients and 33% of survivors. Age 75-85 years (p = 0.018) and hospitalization (p = 0.019) were associated with non-survivor patients. Related to the most common symptoms, the prevalence of fever was similar in both groups, while dyspnea was more frequent among non-survivors and cough among survivors. CONCLUSIONS: This study describes the association of clinical characteristics with the clinical outcomes of survivors and non-survivors of COVID-19 patients, and the specific mutations found in the genome sequences of SARS-CoV-2 in each patient group. Future research on the functional characterization of novel mutations should be performed to understand the role of these variations in SARS-CoV-2 pathogenesis and COVID-19 disease outcomes. These results add new genomic data to better understand the evolution of the SARS-CoV-2 variants that spread in Argentina during the first wave of the COVID-19 pandemic.

Influenza antivirals and animal models.

Influenza A and B viruses are among the most prominent human respiratory pathogens. About 3-5 million severe cases of influenza are associated with 300 000-650 000 deaths per year globally. Antivirals effective at reducing morbidity and mortality are part of the first line of defense against influenza. FDA-approved antiviral drugs currently include adamantanes (rimantadine and amantadine), neuraminidase inhibitors (NAI; peramivir, zanamivir, and oseltamivir), and the PA endonuclease inhibitor (baloxavir). Mutations associated with antiviral resistance are common and highlight the need for further improvement and development of novel anti-influenza drugs. A summary is provided for the current knowledge of the approved influenza antivirals and antivirals strategies under evaluation in clinical trials. Preclinical evaluations of novel compounds effective against influenza in different animal models are also discussed.

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.

Reverse Genetics for Influenza A and B Viruses Driven by Swine Polymerase I Promoter.

Influenza viruses are considered prominent pathogens of humans and animals that are extensively investigated because of public health importance. Plasmid-based reverse genetics is a fundamental tool that facilitates the generation of genetically modified viruses from a cDNA copy. The ability to rescue viruses enables researchers to understand different biological characteristics including IV replication, pathogenesis, and transmission. Furthermore, understanding the biology and ability to manipulate different aspects of the virus can aid in providing a better understanding of the mechanisms of antiviral resistance and development of alternative vaccination strategies. This chapter describes the process of cloning cDNA copies of IAV and IBV RNA segments into a swine polymerase-driven reverse genetics plasmid vector, successful generation of recombinant IVs in swine cells, and propagation of virus in cells or eggs. The swine polymerase reverse genetics system was previously shown to be efficient for de novo rescue of human-, swine-, and avian-origin IAVs and IBV in swine and human origin cell lines utilizing the same protocols discussed in this chapter.

Airborne Transmission of Avian Origin H9N2 Influenza A Viruses in Mammals.

Influenza A viruses (IAV) are widespread viruses affecting avian and mammalian species worldwide. IAVs from avian species can be transmitted to mammals including humans and, thus, they are of inherent pandemic concern. Most of the efforts to understand the pathogenicity and transmission of avian origin IAVs have been focused on H5 and H7 subtypes due to their highly pathogenic phenotype in poultry. However, IAV of the H9 subtype, which circulate endemically in poultry flocks in some regions of the world, have also been associated with cases of zoonotic infections. In this review, we discuss the mammalian transmission of H9N2 and the molecular factors that are thought relevant for this spillover, focusing on the HA segment. Additionally, we discuss factors that have been associated with the ability of these viruses to transmit through the respiratory route in mammalian species. The summarized information shows that minimal amino acid changes in the HA and/or the combination of H9N2 surface genes with internal genes of human influenza viruses are enough for the generation of H9N2 viruses with the ability to transmit via aerosol.

FluB-RAM and FluB-RANS: Genome Rearrangement as Safe and Efficacious Live Attenuated Influenza B Virus Vaccines.

Influenza B virus (IBV) is considered a major respiratory pathogen responsible for seasonal respiratory disease in humans, particularly severe in children and the elderly. Seasonal influenza vaccination is considered the most efficient strategy to prevent and control IBV infections. Live attenuated influenza virus vaccines (LAIVs) are thought to induce both humoral and cellular immune responses by mimicking a natural infection, but their effectiveness has recently come into question. Thus, the opportunity exists to find alternative approaches to improve overall influenza vaccine effectiveness. Two alternative IBV backbones were developed with rearranged genomes, rearranged M (FluB-RAM) and a rearranged NS (FluB-RANS). Both rearranged viruses showed temperature sensitivity in vitro compared with the WT type B/Bris strain, were genetically stable over multiple passages in embryonated chicken eggs and were attenuated in vivo in mice. In a prime-boost regime in naïve mice, both rearranged viruses induced antibodies against HA with hemagglutination inhibition titers considered of protective value. In addition, antibodies against NA and NP were readily detected with potential protective value. Upon lethal IBV challenge, mice previously vaccinated with either FluB-RAM or FluB-RANS were completely protected against clinical disease and mortality. In conclusion, genome re-arrangement renders efficacious LAIV candidates to protect mice against IBV.