The 2009 pandemic H1N1 virus (pH1N1) was derived through reassortment of North American triple reassortant and Eurasian avian-like swine influenza viruses (SIVs). To date, when, how and where the pH1N1 arose is not understood. To investigate viral reassortment, we coinfected cell cultures and a group of pigs with or without preexisting immunity with a Eurasian H1N1 virus, A/Swine/Spain/53207/2004 (SP04), and a North American triple reassortant H1N1 virus, A/Swine/Kansas/77778/2007 (KS07). The infected pigs were cohoused with one or two groups of contact animals to investigate viral transmission. In coinfected MDCK or PK15 continuous cell lines with KS07 and SP04 viruses, more than 20 different reassortant viruses were found. In pigs without or with preexisting immunity (immunized with commercial inactivated swine influenza vaccines) and coinfected with both viruses, six or seven reassortant viruses, as well as the parental viruses, were identified in bronchoalveolar lavage fluid samples from the lungs. Interestingly, only one or two viruses transmitted to and were detected in contact animals. No reassortant containing a gene constellation similar to that of pH1N1 virus was found in either coinfected cells or pigs, indicating that the reassortment event that resulted in the generation of this virus is a rare event that likely involved specific viral strains and/or a favorable, not-yet-understood environment. IMPORTANCE The 2009 pandemic-like H1N1 virus could not be reproduced either in cell cultures or in pigs coinfected with North American triple reassortant H1N1 and Eurasian H1N1 swine influenza viruses. This finding suggests that the generation of the 2009 pandemic H1N1 virus by reassortment was a rare event that likely involved specific viral strains and unknown factors. Different reassortant viruses were detected in coinfected pigs with and without preexisting immunity, indicating that host immunity plays a relevant role in driving viral reassortment of influenza A virus.

Vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were developed in record time and show excellent efficacy and effectiveness against coronavirus disease 2019 (COVID-19). However, currently approved vaccines cannot meet the global demand. In addition, none of the currently used vaccines is administered intranasally to potentially induce mucosal immunity. Here, we tested the safety and immunogenicity of a second-generation SARS-CoV-2 vaccine that includes a stabilized spike antigen and can be administered intranasally. The vaccine is based on a live Newcastle disease virus vector expressing a SARS-CoV-2 spike protein stabilized in a prefusion conformation with six beneficial proline substitutions (AVX/COVID-12-HEXAPRO; Patria). Immunogenicity testing in the pig model showed that both intranasal and intramuscular application of the vaccine as well as a combination of the two induced strong serum neutralizing antibody responses. Furthermore, substantial reactivity to B.1.1.7, B.1.351, and P.1 spike variants was detected. Finally, no adverse reactions were found in the experimental animals at any dose level or delivery route. These results indicate that the experimental vaccine AVX/COVID-12-HEXAPRO (Patria) is safe and highly immunogenic in the pig model. IMPORTANCE Several highly efficacious vaccines for SARS-CoV-2 have been developed and are used in the population. However, the current production capacity cannot meet the global demand. Therefore, additional vaccines-especially ones that can be produced locally and at low cost-are urgently needed. This work describes preclinical testing of a SARS-CoV-2 vaccine candidate which meets these criteria.

The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been identified as the prime target for vaccine development. The spike protein mediates both binding to host cells and membrane fusion and is also so far the only known viral target of neutralizing antibodies. Coronavirus spike proteins are large trimers that are relatively unstable, a feature that might be enhanced by the presence of a polybasic cleavage site in SARS-CoV-2 spike. Exchange of K986 and V987 for prolines has been shown to stabilize the trimers of SARS-CoV-1 and the Middle East respiratory syndrome coronavirus spike proteins. Here, we test multiple versions of a soluble spike protein for their immunogenicity and protective effect against SARS-CoV-2 challenge in a mouse model that transiently expresses human angiotensin-converting enzyme 2 via adenovirus transduction. Variants tested include spike proteins with a deleted polybasic cleavage site, proline mutations, or a combination thereof, besides the wild-type protein. While all versions of the protein were able to induce neutralizing antibodies, only the antigen with both a deleted cleavage site and the K986P and V987P (PP) mutations completely protected from challenge in this mouse model.IMPORTANCE A vaccine for SARS-CoV-2 is urgently needed. A better understanding of antigen design and attributes that vaccine candidates need to have to induce protective immunity is of high importance. The data presented here validate the choice of antigens that contain the PP mutations and suggest that deletion of the polybasic cleavage site may lead to a further-optimized design.

The cytoplasmic helicase RIG-I is an established sensor for viral 5'-triphosphorylated RNA species. Recently, RIG-I was also implicated in the detection of intracellular bacteria. However, little is known about the host cell specificity of this process and the bacterial pathogen-associated molecular pattern (PAMP) that activates RIG-I. Here we show that RNA of Salmonella enterica serovar Typhimurium activates production of beta interferon in a RIG-I-dependent fashion only in nonphagocytic cells. In phagocytic cells, RIG-I is obsolete for detection of Salmonella infection. We further demonstrate that Salmonella mRNA reaches the cytoplasm during infection and is thus accessible for RIG-I. The results from next-generation sequencing analysis of RIG-I-associated RNA suggest that coding bacterial mRNAs represent the activating PAMP. IMPORTANCE S. Typhimurium is a major food-borne pathogen. After fecal-oral transmission, it can infect epithelial cells in the gut as well as immune cells (mainly macrophages, dendritic cells, and M cells). The innate host immune system relies on a growing number of sensors that detect pathogen-associated molecular patterns (PAMPs) to launch a first broad-spectrum response to invading pathogens. Successful detection of a given pathogen depends on colocalization of host sensors and PAMPs as well as potential countermeasures of the pathogen during infection. RIG-I-like helicases were mainly associated with detection of RNA viruses. Our work shows that S. Typhimurium is detected by RIG-I during infection specifically in nonimmune cells.

The capacity of influenza A viruses (IAVs) to host jump from animal reservoir species to humans presents an ongoing pandemic threat. Birds and swine are considered major reservoirs of viral genetic diversity, whereas equines and canines have historically been restricted to one or two stable IAV lineages with no transmission to humans. Here, by sequencing the complete genomes of 16 IAVs obtained from canines in southern China (Guangxi Zhuang Autonomous Region [Guangxi]) in 2013 to 2015, we demonstrate that the evolution of canine influenza viruses (CIVs) in Asian dogs is increasingly complex, presenting a potential threat to humans. First, two reassortant H1N1 virus genotypes were introduced independently from swine into canines in Guangxi, including one genotype associated with a zoonotic infection. The genomes contain segments from three lineages that circulate in swine in China: North American triple reassortant H3N2, Eurasian avian-like H1N1, and pandemic H1N1. Furthermore, the swine-origin H1N1 viruses have transmitted onward in canines and reassorted with the CIV-H3N2 viruses that circulate endemically in Asian dogs, producing three novel reassortant CIV genotypes (H1N1r, H1N2r, and H3N2r [r stands for reassortant]). CIVs from this study were collected primarily from pet dogs presenting with respiratory symptoms at veterinary clinics, but dogs in Guangxi are also raised for meat, and street dogs roam freely, creating a more complex ecosystem for CIV transmission. Further surveillance is greatly needed to understand the full genetic diversity of CIV in southern China, the nature of viral emergence and persistence in the region's diverse canine populations, and the zoonotic risk as the viruses continue to evolve.IMPORTANCE Mammals have emerged as critically underrecognized sources of influenza virus diversity, including pigs that were the source of the 2009 pandemic and bats and bovines that harbor highly divergent viral lineages. Here, we identify two reassortant IAVs that recently host switched from swine to canines in southern China, including one virus with known zoonotic potential. Three additional genotypes were generated via reassortment events in canine hosts, demonstrating the capacity of dogs to serve as "mixing vessels." The continued expansion of IAV diversity in canines with high human contact rates requires enhanced surveillance and ongoing evaluation of emerging pandemic threats.

Highly pathogenic avian influenza A (HPAI) viruses of the H5N1 subtype have recently emerged from avian zoonotic reservoirs to cause fatal human disease. Adaptation of HPAI virus RNA-dependent RNA polymerase (PB1, PB2, and PA proteins) and nucleoprotein (NP) to interactions with mammalian host proteins is thought to contribute to the efficiency of viral RNA synthesis and to disease severity. While proteomics experiments have identified a number of human proteins that associate with H1N1 polymerases and/or viral ribonucleoprotein (vRNP), how these host interactions might regulate influenza virus polymerase functions and host adaptation has been largely unexplored. We took a functional genomics (RNA interference [RNAi]) approach to assess the roles of a network of human proteins interacting with influenza virus polymerase proteins in viral polymerase activity from prototype H1N1 and H5N1 viruses. A majority (18 of 31) of the cellular proteins tested, including RNA-binding (DDX17, DDX5, NPM1, and hnRNPM), stress (PARP1, DDB1, and Ku70/86), and intracellular transport proteins, were required for efficient activity of both H1N1 and H5N1 polymerases. NXP2 and NF90 antagonized both polymerases, and six more RNA-associated proteins exhibited strain-specific phenotypes. Remarkably, 12 proteins differentially regulated H5N1 polymerase according to PB2 genotype at mammalian-adaptive residue 627. Among these, DEAD box RNA helicase DDX17/p72 facilitated efficient human-adapted (627K) H5N1 virus mRNA and viral RNA (vRNA) synthesis in human cells. Likewise, the chicken DDX17 homologue was required for efficient avian (627E) H5N1 infection in chicken DF-1 fibroblasts, suggesting that this conserved virus-host interaction contributes to PB2-dependent host species specificity of influenza virus and ultimately to the outcome of human HPAI infections.

Serum titers of SARS-CoV-2-neutralizing antibodies (nAbs) correlate well with protection from symptomatic COVID-19 but decay rapidly in the months following vaccination or infection. In contrast, measles-protective nAb titers are lifelong after measles vaccination, possibly due to persistence of the live-attenuated virus in lymphoid tissues. We, therefore, sought to generate a live recombinant measles vaccine capable of driving high SARS-CoV-2 nAb responses. Since previous clinical testing of a live measles vaccine encoding a SARS-CoV-2 spike glycoprotein resulted in suboptimal anti-spike antibody titers, our new vectors were designed to encode prefusion-stabilized SARS-CoV-2 spike glycoproteins, trimerized via an inserted peptide domain, and displayed on a dodecahedral miniferritin scaffold. Additionally, to circumvent the blunting of vaccine efficacy by preformed anti-measles antibodies, we extensively modified the measles surface glycoproteins. Comprehensive in vivo mouse testing demonstrated the potent induction of high titer nAbs in measles-immune mice and confirmed the significant contributions to overall potency afforded by prefusion stabilization, trimerization, and miniferritin display of the SARS-CoV-2 spike glycoprotein. In animals primed and boosted with a measles virus (MeV) vaccine encoding the ancestral SARS-CoV-2 spike, high-titer nAb responses against ancestral virus strains were only weakly cross-reactive with the Omicron variant. However, in primed animals that were boosted with a MeV vaccine encoding the Omicron BA.1 spike, antibody titers to both ancestral and Omicron strains were robustly elevated, and the passive transfer of serum from these animals protected K18-ACE2 mice from infection and morbidity after exposure to BA.1 and WA1/2020 strains. Our results demonstrate that by engineering the antigen, we can develop potent measles-based vaccine candidates against SARS-CoV-2.IMPORTANCEAlthough the live-attenuated measles virus (MeV) is one of the safest and most efficacious human vaccines, a measles-vectored COVID-19 vaccine candidate expressing the SARS-CoV-2 spike failed to elicit neutralizing antibody (nAb) responses in a phase-1 clinical trial, especially in measles-immune individuals. Here, we constructed a comprehensive panel of MeV-based COVID-19 vaccine candidates using a MeV with extensive modifications on the envelope glycoproteins (MeV-MR). We show that artificial trimerization of the spike is critical for the induction of nAbs and that their magnitude can be significantly augmented when the spike protein is synchronously fused to a dodecahedral scaffold. Furthermore, preexisting measles immunity did not abolish heterologous immunity elicited by our vector. Our results highlight the importance of antigen optimization in the development of spike-based COVID-19 vaccines and therapies.

An exacerbated immune response is one of the main causes of influenza-induced lung damage during infection. The molecular mechanisms regulating the fate of the initial immune response to infection, either as a protective response or as detrimental immunopathology, are not well understood. The purinergic receptor P2X7 is an ionotropic nucleotide-gated ion channel receptor expressed on immune cells that has been implicated in induction and maintenance of excessive inflammation. Here, we analyze the role of this receptor in a mouse model of influenza virus infection using a receptor knockout (KO) mouse strain. Our results demonstrate that the absence of the P2X7 receptor results in a better outcome to influenza virus infection characterized by reduced weight loss and increased survival upon experimental influenza challenge compared to wild-type mice. This effect was not virus strain specific. Overall lung pathology and apoptosis were reduced in virus-infected KO mice. Production of proinflammatory cytokines and chemokines such as interleukin-10 (IL-10), gamma interferon (IFN-γ), and CC chemokine ligand 2 (CCL2) was also reduced in the lungs of the infected KO mice. Infiltration of neutrophils and depletion of CD11b+ macrophages, characteristic of severe influenza virus infection in mice, were lower in the KO animals. Together, these results demonstrate that activation of the P2X7 receptor is involved in the exacerbated immune response observed during influenza virus infection.IMPORTANCE A hallmark of influenza virus infection is the development of lung pathology induced by an exacerbated immune response. The mechanisms shared by the antiviral host defense required for viral clearance and those required for development of immunopathology are not clearly understood. Purinergic receptors, and in particular the purinergic receptor P2X7 (P2X7r), are involved in activation of the immune response. We used mice lacking the P2X7r (P2X7r KO mice) to better understand the mechanisms that lead to development of lung pathology during influenza virus infection. In our studies, we observed that P2X7r KO mice developed less lung immunopathology and had better survival than the wild-type mice. These results implicate P2X7r in the induction of an exacerbated local immune response to influenza virus and help us to better understand the mechanisms leading to the lung immunopathology observed during severe viral infections.

Although highly effective in the general population when well matched to circulating influenza virus strains, current influenza vaccines are limited in their utility due to the narrow breadth of protection they provide. The strain specificity of vaccines presently in use mirrors the exquisite specificity of the neutralizing antibodies that they induce, that is, antibodies which bind to the highly variable globular head domain of hemagglutinin (HA). Herein, we describe the construction of a novel immunogen comprising the conserved influenza HA stalk domain and lacking the globular head. Vaccination of mice with this headless HA construct elicited immune sera with broader reactivity than those obtained from mice immunized with a full-length HA. Furthermore, the headless HA vaccine provided full protection against death and partial protection against disease following lethal viral challenge. Our results suggest that the response induced by headless HA vaccines is sufficiently potent to warrant their further development toward a universal influenza virus vaccine.

Ebola virus (EBOV) protein VP35 inhibits production of interferon alpha/beta (IFN) by blocking RIG-I-like receptor signaling pathways, thereby promoting virus replication and pathogenesis. A high-throughput screening assay, developed to identify compounds that either inhibit or bypass VP35 IFN-antagonist function, identified five DNA intercalators as reproducible hits from a library of bioactive compounds. Four, including doxorubicin and daunorubicin, are anthracycline antibiotics that inhibit topoisomerase II and are used clinically as chemotherapeutic drugs. These compounds were demonstrated to induce IFN responses in an ATM kinase-dependent manner and to also trigger the DNA-sensing cGAS-STING pathway of IFN induction. These compounds also suppress EBOV replication in vitro and induce IFN in the presence of IFN-antagonist proteins from multiple negative-sense RNA viruses. These findings provide new insights into signaling pathways activated by important chemotherapy drugs and identify a novel therapeutic approach for IFN induction that may be exploited to inhibit RNA virus replication.IMPORTANCE Ebola virus and other emerging RNA viruses are significant but unpredictable public health threats. Therapeutic approaches with broad-spectrum activity could provide an attractive response to such infections. We describe a novel assay that can identify small molecules that overcome Ebola virus-encoded innate immune evasion mechanisms. This assay identified as hits cancer chemotherapeutic drugs, including doxorubicin. Follow-up studies provide new insight into how doxorubicin induces interferon (IFN) responses, revealing activation of both the DNA damage response kinase ATM and the DNA sensor cGAS and its partner signaling protein STING. The studies further demonstrate that the ATM and cGAS-STING pathways of IFN induction are a point of vulnerability not only for Ebola virus but for other RNA viruses as well, because viral innate immune antagonists consistently fail to block these signals. These studies thereby define a novel avenue for therapeutic intervention against emerging RNA viruses.

Ebola virus (EBOV) VP24 protein is a nucleocapsid-associated protein that inhibits interferon (IFN) gene expression and counteracts the IFN-mediated antiviral response, preventing nuclear import of signal transducer and activator of transcription 1 (STAT1). Proteomic studies to identify additional EBOV VP24 partners have pointed to the nuclear membrane component emerin as a potential element of the VP24 cellular interactome. Here, we have further studied this interaction and its impact on cell biology. We demonstrate that VP24 interacts with emerin but also with other components of the inner nuclear membrane, such as lamin A/C and lamin B. We also show that VP24 diminishes the interaction between emerin and lamin A/C and compromises the integrity of the nuclear membrane. This disruption is associated with nuclear morphological abnormalities, activation of a DNA damage response, the phosphorylation of extracellular signal-regulated kinase (ERK), and the induction of interferon-stimulated gene 15 (ISG15). Interestingly, expression of VP24 also promoted the cytoplasmic translocation and downmodulation of barrier-to-autointegration factor (BAF), a common interactor of lamin A/C and emerin, leading to repression of the BAF-regulated CSF1 gene. Importantly, we found that EBOV infection results in the activation of pathways associated with nuclear envelope damage, consistent with our observations in cells expressing VP24. In summary, here we demonstrate that VP24 acts at the nuclear membrane, causing morphological and functional changes in cells that recapitulate several of the hallmarks of laminopathy diseases. IMPORTANCE The Ebola virus (EBOV) VP24 protein is a nucleocapsid-associated protein with multiple functions. Proteomic studies have identified the cellular nuclear membrane component emerin as a potential VP24 interactor. Here, we demonstrate that VP24 not only interacts with emerin but also with lamin A/C and lamin B, prompting nuclear membrane disruption. This disruption is associated with nuclear morphological abnormalities, activation of a DNA damage response, the phosphorylation of extracellular signal-regulated kinase (ERK), and the induction of interferon-stimulated gene 15 (ISG15). Interestingly, VP24 also promotes the cytoplasmic translocation and downmodulation of barrier-to-autointegration factor (BAF), leading to repression of the BAF-regulated CSF1 gene. Finally, we show that EBOV infection also results in the activation of pathways associated with nuclear envelope damage, consistent with our observations in cells expressing VP24. These results reveal novel activities of EBOV VP24 protein, resulting in a cell phenotype similar to that of most laminopathies, with potential impact on EBOV replication.