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Influenza A viruses (IAVs) are the causative agents of one of the most important viral respiratory diseases in pigs and humans. Human and swine IAV are prone to interspecies transmission, leading to regular incursions from human to pig and vice versa. This bidirectional transmission of IAV has heavily influenced the evolutionary history of IAV in both species. Transmission of distinct human seasonal lineages to pigs, followed by sustained within-host transmission and rapid adaptation and evolution, represent a considerable challenge for pig health and production. Consequently, although only subtypes of H1N1, H1N2, and H3N2 are endemic in swine around the world, extensive diversity can be found in the hemagglutinin (HA) and neuraminidase (NA) genes, as well as the remaining six genes. We review the complicated global epidemiology of IAV in swine and the inextricably entangled implications for public health and influenza pandemic planning.

Tuberculosis (TB) is the leading infectious cause of death among people living with HIV. People living with HIV are more susceptible to contracting Mycobacterium tuberculosis and often have worsened TB disease. Understanding the immunologic defects caused by HIV and the consequences it has on M. tuberculosis coinfection is critical in combating this global health epidemic. We previously showed in a model of SIV and M. tuberculosis coinfection in Mauritian cynomolgus macaques (MCM) that SIV/M. tuberculosis–coinfected MCM had rapidly progressive TB. We hypothesized that pre-existing SIV infection impairs early T cell responses to M. tuberculosis infection. We infected MCM with SIVmac239, followed by coinfection with M. tuberculosis Erdman 6 mo later. Although similar, TB progression was observed in both SIV+ and SIV-naive animals at 6 wk post–M. tuberculosis infection; longitudinal sampling of the blood (PBMC) and airways (bronchoalveolar lavage) revealed a significant reduction in circulating CD4+ T cells and an influx of CD8+ T cells in airways of SIV+ animals. At sites of M. tuberculosis infection (i.e., granulomas), SIV/M. tuberculosis–coinfected animals had a higher proportion of CD4+ and CD8+ T cells expressing PD-1 and TIGIT. In addition, there were fewer TNF-producing CD4+ T cells in granulomas of SIV/M. tuberculosis–coinfected animals. Taken together, we show that concurrent SIV infection alters T cell phenotypes in granulomas during the early stages of TB disease. As it is critical to establish control of M. tuberculosis replication soon postinfection, these phenotypic changes may distinguish the immune dysfunction that arises from pre-existing SIV infection, which promotes TB progression.

Avian influenza virus (AIV) H9N2 subtype is an infectious pathogen that can affect both the respiratory and gastrointestinal systems in chickens and continues to have an important economic impact on the poultry industry. While the host innate immune response provides control of virus replication in early infection, the adaptive immune response aids to clear infections and prevent future invasion. Modelling virus-innate immune response pathways can improve our understanding of early infection dynamics and help to guide our understanding of virus shedding dynamics that could lead to reduced transmission between hosts. While some countries use vaccines for the prevention of H9N2 AIV in poultry, the virus continues to be endemic in regions of Eurasia and Africa, indicating a need for improved vaccine efficacy or vaccination strategies. Here we explored how three type-I interferon (IFN) pathways affect respiratory virus shedding patterns in infected chickens using a within-host model. Additionally, prime and boost vaccination strategies for a candidate H9N2 AIV vaccine are assessed for the ability to elicit seroprotective antibody titres. The model demonstrates that inclusion of virus sensitivity to intracellular type-I IFN pathways results in a shedding pattern most consistent with virus titres observed in infected chickens, and the inclusion of a cellular latent period does not improve model fit. Furthermore, early administration of a booster dose two weeks after the initial vaccine is administered results in seroprotective titres for the greatest length of time for both broilers and layers. These results demonstrate that type-I IFN intracellular mechanisms are required in a model of respiratory virus shedding in H9N2 AIV infected chickens, and also highlights the need for improved vaccination strategies for laying hens.

In 2012, swine influenza surveillance detected a novel reassorted influenza A virus (IAV) strain containing human-seasonal hemagglutinin (HA) and neuraminidase (NA). Subsequently, these viruses reassorted, maintaining only the human-origin H3, which resulted in a new lineage of viruses that became the most frequently detected H3 clade in US swine (2010.1 HA clade). Here, we assessed the antigenic phenotype, virulence, and transmission characteristics of this virus lineage following its introduction to swine. Relative to 2010.1 viruses from 2012 and 2014, recent 2010.1 contemporary strains from 2015 to 2017 resulted in equivalent macroscopic lung lesions and transmission in pigs. A single mutation at amino acid residue 145 within the previously defined HA antigenic motif was associated with a change of antigenic phenotype, potentially impairing vaccine efficacy. Contemporary 2010.1 viruses circulating in swine since 2012 were significantly different from both pre-2012H3N2 in swine and human-seasonal H3N2 viruses and demonstrated continued evolution within the lineage.

Malaria remains one of the most deadly infectious diseases, causing hundreds of thousands of deaths each year, primarily in young children and pregnant mothers. Here, we report the discovery and derivatization of a series of pyrazolo[3,4-b]pyridines targeting Plasmodium falciparum, the deadliest species of the malaria parasite. Hit compounds in this series display sub-micromolar in vitro activity against the intraerythrocytic stage of the parasite as well as little to no toxicity against the human fibroblast BJ and liver HepG2 cell lines. In addition, our hit compounds show good activity against the liver stage of the parasite but little activity against the gametocyte stage. Parasitological profiles, including rate of killing, docking, and molecular dynamics studies, suggest that our compounds may target the Qo binding site of cytochrome bc1.

Most H1 influenza A viruses (IAVs) of swine are derived from past human viruses. As human population immunity against these IAVs gradually decreases, the risk of reintroduction to humans increases. We examined 549 serum samples from persons 0-97 years of age collected in Belgium during 2017-2018 for hemagglutination inhibiting and virus neutralizing antibodies against 7 major H1 swine IAV (swIAV) clades and 3 human progenitor IAVs. Seroprevalence (titers >= 40) rates were >= 50% for classical swine and European human-like swIAVs, >= 24% for North American human-like delta 1a and Asian avian-like swIAVs, and

Human-to-swine transmission of influenza A virus (IAV) repeatedly occurs, leading to sustained transmission and increased diversity in swine; human seasonal H3N2 introductions occurred in the 1990s and 2010s and were maintained in North American swine. Swine H3N2 strains were subsequently associated with zoonotic infections, highlighting the need to understand the risk of endemic swine IAV to humans. We quantified antigenic distances between swine H3N2 and human seasonal vaccine strains from 1973 to 2014 using a panel of monovalent antisera raised in pigs in hemagglutination inhibition (HI) assays. Swine H3N2 lineages retained the closest antigenic similarity to human vaccine strains from the decade of incursion. Swine lineages from the 1990s were antigenically more similar to human vaccine strains of the mid-1990s but had substantial distance from recent human vaccine strains. In contrast, lineages from the 2010s were closer to human vaccine strains from 2011 and 2014 and the most antigenically distant from human vaccine strains prior to 2007. HI assays using ferret antisera demonstrated that swine lineages from the 1990s and 2010s had significant fold reductions compared to the homologous HI titer of the nearest pandemic preparedness candidate vaccine virus (CVV) or seasonal vaccine strain. The assessment of postinfection and postvaccination human serum cohorts demonstrated limited cross-reactivity to swine H3N2 from the 1990s, especially in older adults born before the 1970s. We identified swine strains to which humans are likely to lack population immunity or are not protected against by a current human seasonal vaccine or CVV to use in prioritizing future human CVV strain selection. IMPORTANCE Human H3N2 influenza A viruses spread to pigs in North America in the 1990s and more recently in the 2010s. These cross-species events led to sustained circulation and increased H3N2 diversity in pig populations. The evolution of H3N2 in swine led to a reduced similarity to human seasonal H3N2 and the vaccine strains used to protect human populations. We quantified the antigenic phenotypes and found that North American swine H3N2 lineages retained more antigenic similarity to historical human vaccine strains from the decade of incursion but had substantial differences compared to recent human vaccine strains. Additionally, pandemic preparedness vaccine strains demonstrated a loss of similarity to contemporary swine strains. Finally, human sera revealed that although these adults had antibodies against human H3N2 strains, many had limited immunity to swine H3N2, especially older adults born before 1970. Antigenic assessment of swine H3N2 provides critical information for pandemic preparedness and candidate vaccine development.

The neuraminidase (NA) and hemagglutinin (HA) are essential surface glycoproteins of influenza A virus (IAV). In this study, the evolution of subtype N2 NA paired with H1 and H3 subtype HA in swine was evaluated to understand if the genetic diversity of HA and NA were linked. Using time-scaled Bayesian phylodynamic analyses, the relationships of paired swine N2 with H1 or H3 from 2009 to 2018 were evaluated. These data demonstrated increased relative genetic diversity within the major N2 clades circulating in swine in the USA (N2.1998 between 2014 and 2017 and N2.2002 between 2010 and 2016). Preferential pairing was observed among specific NA and HA genetic clades. Gene reassortment between cocirculating influenza A strains resulted in novel pairings that persisted. The changes in genetic diversity in the NA gene were quantified using Bayesian phylodynamic analyses, and increases in diversity were observed subsequent to novel NA–HA reassortment events. The rate of evolution among NA–N2 clades and HA–H1 and HA–H3 clades were similar. Bayesian phylodynamic analyses demonstrated strong spatial patterns in N2 genetic diversity, but frequent interstate movement of rare N2 clades provided opportunity for reassortment and emergence of new N2–HA pairings. The frequent regional movement of pigs and their influenza viruses is an explanation for the documented patterns of reassortment and subsequent changes in gene diversity. The reassortment and evolution of NA and linked HA evolution may result in antigenic drift of both major surface glycoproteins, reducing vaccine efficacy, with subsequent impact on animal health.

Rift Valley Fever Virus (RVFV) is a zoonotic pathogen with pandemic potential. RVFV entry is mediated by the viral glycoprotein (Gn), but host entry factors remain poorly defined. Our genome-wide CRISPR screen identified low-density lipoprotein receptor-related protein 1 (mouse Lrp1/human LRP1), heat shock protein (Grp94), and receptor associated protein (RAP) as critical host factors for RVFV infection. RVFV Gn directly binds to specific clusters Lrp1 and is glycosylation independent. Exogenous addition of murine RAP domain 3 (mRAP(D3)) and anti-Lrp1 antibodies neutralize RVFV infection in taxonomically diverse cell lines. Mice treated with mRAP(D3) and infected with pathogenic RVFV are protected from disease and death. A mutant mRAPD3 that binds Lrp1 weakly failed to protect from RVFV infection. Altogether, these data support Lrp1 as a host entry factor for RVFV infection and defines a new target to limit RVFV infections.

Venezuelan equine encephalitis virus (VEEV) is a positively-stranded RNA arbovirus of the genus Alphavirus that causes encephalitis in humans. Cynomolgus macaques are a relevant model of the human disease caused by VEEV and are useful in exploring pathogenic mechanisms and the host response to VEEV infection. Macaques were exposed to small-particle aerosols containing virus derived from an infectious clone of VEEV strain INH-9813, a subtype IC strain isolated from a human infection. VEEV-exposed macaques developed a biphasic fever after infection similar to that seen in humans. Maximum temperature deviation correlated with the inhaled dose, but fever duration did not. Neurological signs, suggestive of virus penetration into the CNS, were predominantly seen in the second febrile period. Electroencephalography data indicated a statistically significant decrease in all power bands and circadian index during the second febrile period that returned to normal after fever resolved. Intracranial pressure increased late in the second febrile period. On day 6 post-infection macaques had high levels of MCP-1 and IP-10 chemokines in the CNS, as well as a marked increase of T lymphocytes and activated microglia. More than four weeks after infection, viral genomic RNA was found in the brain, cerebrospinal fluid and cervical lymph nodes. Pro-inflammatory cytokines & chemokines, infiltrating leukocytes and pathological changes were seen in the CNS tissues of macaques euthanized at these times. These data are consistent with persistence of virus replication and/or genomic RNA and potentially, inflammatory sequelae in the central nervous system after resolution of acute VEEV disease.

Two separate introductions of human seasonal N2 neuraminidase genes were sustained in United States swine since 1998 (N2-98) and 2002 (N2-02). Herein, we characterized the antigenic evolution of the N2 of swine influenza A virus (IAV) across two decades following each introduction. The N2-98 and N2-02 expanded in genetic diversity, with two statistically supported monophyletic clades within each lineage. To assess antigenic drift in swine N2 following the human-to-swine spillover events, we generated a panel of swine N2 antisera against representative N2 and quantified the antigenic distance between wild-type viruses using enzyme-linked lectin assay and antigenic cartography. The antigenic distance between swine and human N2 was smallest between human N2 circulating at the time of each introduction and the archetypal swine N2. However, sustained circulation and evolution in swine of the two N2 lineages resulted in significant antigenic drift, and the N2-98 and N2-02 swine N2 lineages were antigenically distinct. Although intra-lineage antigenic diversity was observed, the magnitude of antigenic drift did not consistently correlate with the observed genetic differences. These data represent the first quantification of the antigenic diversity of neuraminidase of IAV in swine and demonstrated significant antigenic drift from contemporary human seasonal strains as well as antigenic variation among N2 detected in swine. These data suggest that antigenic mismatch may occur between circulating swine IAV and vaccine strains. Consequently, consideration of the diversity of N2 in swine IAV for vaccine selection may likely result in more effective control, and aid public health initiatives for pandemic preparedness. Importance Antibodies inhibiting the neuraminidase (NA) of influenza A virus (IAV) reduce clinical disease, virus shedding, and transmission, particularly in the absence of neutralizing immunity against the hemagglutinin. To understand antibody recognition of the genetically diverse NA in U.S. swine IAV, we characterized the antigenic diversity of N2 from swine and humans. N2 detected in swine IAV were derived from two distinct human-to-swine spillovers that persisted, are antigenically distinct, and underwent antigenic drift. These findings highlight the need for continued surveillance and vaccine development in swine with increased focus on the NA. Additionally, human seasonal N2 isolated after 2005 were poorly inhibited by representative swine N2 antisera, suggesting a lack of cross-reactive NA antibody mediated immunity between contemporary swine and human N2. Bidirectional transmission between humans and swine represents a One Health challenge, and determining the correlates of immunity to emerging IAV strains is critical to mitigate zoonotic and reverse-zoonotic transmission.

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, introduction of genes encoding immunomodulatory functions into a candidate LAIV that can serve as natural adjuvants to improve overall vaccine safety and efficacy.

Pyrethroid insecticides are widely used to control mosquitoes that transmit diseases such as West Nile virus (WNV) to humans. A single nucleotide polymorphism (SNP) in the knockdown resistance locus (kdr) of the voltage gated sodium channel (Vgsc) gene of Culex mosquitoes confers knockdown resistance to pyrethroids. PCR-based assays that detect these SNPs in Culex species are currently available for Culex pipiens Linnaeus and Culex quinquefasciatus Say. RNAseq was employed to sequence the coding region of Vgsc for Culex tarsalis Coquillett and Culex erythrothorax Dyar, two WNV vectors. We utilized the cDNA sequence to develop a quantitative reverse transcriptase PCR assay that detects the L1014F mutation in the kdr of Vgsc. Because this locus is conserved, the assay successfully detected the SNPs in multiple Culex spp. vectors of WNV in the United States. The resulting Culex RTkdr assay was validated using quantitative PCR, CDC bottle bioassays, and sequencing of PCR products. Using sequencing, we determined the accuracy of the Culex RTkdr assay was 99%. Pyrethroid resistance was more common among Cx. pipiens than other Culex spp. and co-occured with agriculture. We anticipate that public health and vector control agencies may utilize the Culex RTkdr assay to map the distribution of pyrethroid resistance in Culex species to more efficiently control mosquitoes and the diseases they transmit.

Malaria remains one of the deadliest diseases on the planet, infecting an estimated 229 million individuals in 2019 with more than 400,000 associated deaths, primarily in young children and pregnant women. 1 Malaria is caused by Plasmodium parasite infection of the liver and blood, with P. falciparum accounting for the vast majority of deaths. 2 The complexity of the Plasmodium life cycle3 coupled with mounting resistance to current treatments4 underscores the need for new treatment options. As a part of this discovery effort, we recently identified a number of active compounds in a high-throughput screen designed to identify molecules that arrest the growth of P. falciparum in co-culture with human erythrocytes. 5 One compound displaying notable activity was compound 1, a 2,6-dibenzylidenecyclohexanone shown in.Fig 1

Individuals infected with both HIV and Mycobacterium tuberculosis (Mtb) are more likely to develop severe Tuberculosis (TB) disease than HIV-naïve individuals. To understand how a chronic pre-existing Simian immunodeficiency virus (SIV) infection impairs the early immune response to Mtb, we used the Mauritian cynomolgus macaque (MCM) model of SIV/Mtb co-infection. We examined the relationship between peripheral viral control and Mtb burden, Mtb dissemination, and immunological function between SIV+ spontaneous controllers, SIV+ non-controllers, and SIV-naïve MCM who were challenged with a barcoded Mtb Erdman strain and necropsied six weeks post infection. Mycobacterial burden was highest in the SIV+ non-controllers in all assessed tissues. In lung granulomas, we found the frequency of CD4+ T cells producing TNFα was reduced in all SIV+ MCM, but CD4+ T cells producing IFNγ were only lower in the SIV+ non-controllers. Further, while all SIV+ MCM had more PD1+ and TIGIT+ T cells in the lung granulomas relative to SIV-naïve MCM, SIV+ controllers exhibited the highest frequency of cells expressing these markers. To measure the effect of SIV infection on within-host bacterial dissemination, we sequenced the molecular barcodes of Mtb present in each tissue and characterized the complexity of the Mtb populations. While Mtb population complexity was not associated with infection group, lymph nodes had increased complexity when compared to lung granulomas across all groups. These results provide evidence SIV+ animals, independent of viral control, exhibit dysregulated immune responses and enhanced dissemination of Mtb, likely contributing to the poor TB disease course across all SIV/Mtb co-infected animals.ImportanceHIV and TB remain significant global health issues, despite the availability of treatments. Individuals with HIV, including those who are virally suppressed, are at an increased risk to develop and succumb to severe TB disease when compared to HIV-naïve individuals. Our study aims to understand the relationship between SIV replication, mycobacterial growth, and immunological function in the tissues of co-infected Mauritian cynomolgus macaques during the early phase of Mtb infection. Here we demonstrate that increased viral replication is associated with increased bacterial burden in the tissues and impaired immunologic responses, and that the damage attributed to virus infection is not fully eliminated when animals spontaneously control virus replication.

The potential for emerging mosquito-borne viruses to cause fetal infection in pregnant women was overlooked until the Zika fever outbreak several years ago. Rift Valley fever virus (RVFV) is an emerging arbovirus with a long history of fetal infection and death in pregnant livestock. The effect of RVFV infection on pregnant women is not well understood. This Gem examines the effects that this important emerging pathogen has during pregnancy, its potential impact on pregnant women, and the current research efforts designed to understand and mitigate adverse effects of RVFV infection during pregnancy.

The genetic diversity of influenza A viruses circulating in swine in Mexico complicates control efforts in animals and presents a threat to humans, as shown by influenza A(H1N1)pdm09 virus. To describe evolution of swine influenza A viruses in Mexico and evaluate strains for vaccine development, we sequenced the genomes of 59 viruses and performed antigenic cartography on strains from 5 regions. We found that genetic and antigenic diversity were particularly high in southeast Mexico because of repeated introductions of viruses from humans and swine in other regions in Mexico. We identified novel reassortant H3N2 viruses with genome segments derived from 2 different viruses that were independently introduced from humans into swine: pandemic H1N1 viruses and seasonal H3N2 viruses. The Mexico swine viruses are antigenically distinct from US swine lineages. Protection against these viruses is unlikely to be afforded by US virus vaccines and would require development of new vaccines specifically targeting these diverse strains.

Influenza A virus in swine (IAV-S) is an important pathogen in pigs in the United States, in addition to posing a potential risk to humans through zoonotic events. Intervention strategies continue to be explored to better control virus circulation. Improved surveillance efforts has led to significantly increased sequence data available on circulating strains, vastly improving our understanding of the genetic and antigenic diversity of IAV-S. IAV-S in North America is characterized by repeated spillover events of human viruses into pigs followed by genetic and antigenic diversification. An important gap that needs to be addressed is our understanding of the role that various vaccine platforms have on efficacy against antigenically heterologous challenge. Currently licensed vaccines often update their components to adapt to a dynamic antigenic landscape and newly developed technologies are continue to be approved. Hence, it remains critical to test the performance of vaccines against challenge with antigenically distinct viruses. We tested the level of protection conferred by an alphavirus-vectored hemagglutinin (HA) subunit vaccine, delivered as a monovalent or bivalent formulation, against challenge with IAV-S. Monovalent alphavirus-vectored HA vaccines provided efficient protection against challenge with viruses with matched and mismatched HA, although in one mismatched HA challenge group there was a trend for reduced protection. A bivalent vaccine, in which two HA’s were simultaneously delivered, was effective in producing antibody response against both antigens and provided protection against challenge. The alphavirus platform is a promising new technology available to swine producers to help reduce the burden of disease caused by IAV-S.

In 2017, outbreaks of low and highly pathogenic avian H7N9 viruses were reported in four States in the United States. In total, over 270 000 birds died or were culled, causing significant economic loss. The potential for avian-to-swine transmission of the U.S. avian H7N9 was unknown. In an experimental challenge in swine using a representative low pathogenic H7N9 (A/chicken/Tennessee/17-007431-3/2017; LPAI TN/17) isolated from these events, no infectious virus in the upper and minimal virus in the lower respiratory tract was detected, nor was lung pathology or evidence of transmission in pigs observed, indicating that the virus cannot efficiently infect swine.

Within host-parasite communities, viral co-circulation and co-infections of hosts are the norm, yet studies of significant emerging zoonoses tend to focus on a single parasite species within the host. Using a multiplexed paramyxovirus bead-based PCR on urine samples from Australian flying foxes, we show that multi-viral shedding from flying fox populations is common. We detected up to nine bat paramyxoviruses shed synchronously. Multi-viral shedding infrequently coalesced into an extreme, brief and spatially restricted shedding pulse, coinciding with peak spillover of Hendra virus, an emerging fatal zoonotic pathogen of high interest. Such extreme pulses of multi-viral shedding could easily be missed during routine surveillance yet have potentially serious consequences for spillover of novel pathogens to humans and domestic animal hosts. We also detected co-occurrence patterns suggestive of the presence of interactions among viruses, such as facilitation and cross-immunity. We propose that multiple viruses may be interacting, influencing the shedding and spillover of zoonotic pathogens. Understanding these interactions in the context of broader scale drivers, such as habitat loss, may help predict shedding pulses of Hendra virus and other fatal zoonoses.

Influenza A virus (IAV) causes a disease burden in the swine industry in the US and is a challenge to prevent due to substantial genetic and antigenic diversity of IAV that circulate in pig populations. Whole inactivated virus (WIV) vaccines formulated with oil-in-water (OW) adjuvant are commonly used in swine. However, WIV-OW are associated with vaccine-associated enhanced respiratory disease (VAERD) when the hemagglutinin and neuraminidase of the vaccine strain are mismatched with the challenge virus. Here, we assessed if different types of adjuvant in WIV vaccine formulations impacted VAERD outcome. WIV vaccines with a swine δ1-H1N2 were formulated with different commercial adjuvants: OW1, OW2, nano-emulsion squalene-based (NE) and gel polymer (GP). Pigs were vaccinated twice by the intramuscular route, 3 weeks apart, then challenged with an H1N1pdm09 three weeks post-boost and necropsied at 5 days post infection. All WIV vaccines elicited antibodies detected using the hemagglutination inhibition (HI) assay against the homologous vaccine virus, but not against the heterologous challenge virus; in contrast, all vaccinated groups had cross-reactive IgG antibody and IFN-γ responses against H1N1pdm09, with a higher magnitude observed in OW groups. Both OW groups demonstrated robust homologous HI titers and cross-reactivity against heterologous H1 viruses in the same genetic lineage. However, both OW groups had severe immunopathology consistent with VAERD after challenge when compared to NE, GP, and non-vaccinated challenge controls. None of the WIV formulations protected pigs from heterologous virus replication in the lungs or nasal cavity. Thus, although the type of adjuvant in the WIV formulation played a significant role in the magnitude of immune response to homologous and antigenically similar H1, none tested here increased the breadth of protection against the antigenically-distinct challenge virus, and some impacted immunopathology after challenge.

Ehrlichiosis, caused by Gram-negative bacteria of the genus Ehrlichia , is considered an emerging infectious disease due to the increasing number of reported cases. Symptoms are non-specific and occur within 1 to 2 weeks following the bite of an infected tick. Confirmatory laboratory diagnostic methods vary in sensitivity and specimen requirements, which can lead to delayed diagnosis. PCR testing serves as an efficient approach to Ehrlichia confirmation in the acute stage of illness. Published assays have been effectively used to detect human ehrlichiosis at limit of detections ranging from 10 to 50 genomic copies (GC) of Ehrlichia DNA. With the discovery of new species capable of human infection, we wanted to develop assays that are sensitive and encompass a wide range of Ehrlichia. Here we developed and validated two sensitive and specific real-time PCR assays (PanE1 and PanE2) for the detection of Ehrlichia species, as well as two real-time PCR assays (ECh2 and ECh4) for the detection of Ehrlichia chaffeensis , specifically. The limit of detection was determined to be 10 GC per reaction with 100% confidence, and as little as 1 GC with lower efficiencies. Accuracy was assessed at 100% correlation. Specificity from exclusivity testing demonstrated that neither the Ehrlichia species assays (n = 60), nor the E. chaffeensis specific assays (n = 64) had cross reactivity with near neighbors or environmental bacteria. A positive predictive value of 100% and a negative predictive value of ≥93% was determined by evaluating banked clinical specimens from 62 patients with the assays. These real-time PCR assays are effective tools to detect human Ehrlichia species during the acute stage of illness. Early detection of Ehrlichia infection by these real-time PCR assays can facilitate diagnosis and treatment.

Human-to-swine transmission of seasonal influenza viruses has led to sustained human-like influenza viruses circulating in the U.S. swine population. While some reverse zoonotic-origin viruses adapt and become enzootic in swine, nascent reverse zoonoses may result in virus detections that are difficult to classify as “swine-origin” or “human-origin” due to the genetic similarity of circulating viruses. This is the case for human-origin influenza A(H1N1) pandemic 2009 (pdm09) viruses detected in pigs following numerous reverse zoonosis events since the 2009 pandemic. We report the identification of two human infections with A(H1N1)pdm09 viruses originating from swine hosts and classify them as “swine-origin” variant influenza viruses based on phylogenetic analysis and sequence comparison methods. Phylogenetic analyses of viral genomes from two cases revealed these viruses were reassortants containing A(H1N1)pdm09 hemagglutinin (HA) and neuraminidase (NA) genes with genetic combinations derived from the triple reassortant internal gene cassette. Follow-up investigations determined that one individual had direct exposure to swine in the week preceding illness onset, while another did not report swine exposure. The swine-origin A(H1N1) variant cases were resolved by full genome sequence comparison of the variant viruses to swine influenza genomes. However, if reassortment does not result in the acquisition of swine-associated genes and swine virus genomic sequences are not available from the exposure source, future cases may not be discernible. We have developed a pipeline that performs maximum likelihood analyses, a k-mer-based set difference algorithm, and random forest algorithms to identify swine-associated sequences in the hemagglutinin gene to differentiate between human-origin and swine-origin A(H1N1)pdm09 viruses. IMPORTANCE Influenza virus infects a wide range of hosts, resulting in illnesses that vary from asymptomatic cases to severe pneumonia and death. Viral transfer can occur between human and nonhuman hosts, resulting in human and nonhuman origin viruses circulating in novel hosts. In this work, we have identified the first case of a swine-origin influenza A(H1N1)pdm09 virus resulting in a human infection. This shows that these viruses not only circulate in swine hosts, but are continuing to evolve and distinguish themselves from previously circulating human-origin influenza viruses. The development of techniques for distinguishing human-origin and swine-origin viruses are necessary for the continued surveillance of influenza viruses. We show that unique genetic signatures can differentiate circulating swine-associated strains from circulating human-associated strains of influenza A(H1N1)pdm09, and these signatures can be used to enhance surveillance of swine-origin influenza.

Tuberculosis (TB) is the leading infectious cause of death among people living with HIV (PLHIV). PLHIV are more susceptible to contracting Mycobacterium tuberculosis (Mtb) infection and often have worsened TB disease. Understanding the immunologic defects caused by HIV and the consequences it has on Mtb co-infection is critical in combating this global health epidemic. We previously established a model of simian immunodeficiency virus (SIV) and Mtb co-infection in Mauritian cynomolgus macaques (MCM), and showed that SIV/Mtb co-infected MCM had rapidly progressive TB. We hypothesized that pre-existing SIV infection impairs early T cell responses to Mtb infection. To test our hypothesis, we infected MCM with SIVmac239 intrarectally followed by co-infection with a low dose of Mtb Erdman 6 months later. SIV-naïve MCM were infected with Mtb alone as controls. Six weeks after Mtb infection, animals were necropsied and immune responses were measured by multiparameter flow cytometry. While the two groups exhibited similar TB progression at time of necropsy (Nx), longitudinal sampling of the blood (PBMC) and airways (BAL) revealed a significant reduction in circulating CD4+ T cells and an influx of CD8+ T cells in airways following Mtb co-infection of SIV+ animals. Differences in the activation markers CD69, PD-1, and TIGIT were observed. At sites of Mtb infection (i.e. granulomas), SIV/Mtb co-infected animals had a higher proportion of CD4+ and CD8+ T cells expressing PD-1 and TIGIT. In addition, there were fewer TNF-producing CD4+ and CD8+ T cells in granulomas and airways of SIV/Mtb co-infected animals. Taken together, we show that concurrent SIV infection alters T cell phenotypes in granulomas during the early stages of TB disease. As it is critical to establish control of Mtb replication soon after infection, these phenotypic changes may distinguish the immune dysfunction that arises from pre-existing SIV infection which promotes TB progression.Author SummaryPeople living with HIV are incredibly susceptible to TB and, when co-infected with Mtb, often develop serious TB disease. We do not yet understand precisely how HIV infection impairs the early stages of the adaptive immune response against Mtb bacilli. We employed a non-human primate model of HIV, using SIV as a surrogate for HIV, followed by Mtb co-infection to investigate the immunologic defects associated with pre-existing SIV infection over the first six weeks of Mtb co-infection. Our study focused on CD4+ and CD8+ T cells as these cells are known to play an important role in Mtb control. We found more CD8+ T cells in granulomas, the sites of Mtb infection, from SIV/Mtb co-infected animals, with little difference in CD4+ T cells. SIV/Mtb co-infected animals and animals infected with SIV alone had a higher proportion of both CD4+ and CD8+ T cells expressing activation markers compared to SIV-naïve animals, consistent with SIV-dependent immune activation. Notably, we observed a lower proportion of TNF-producing T cells, a cytokine critical for Mtb control, in granulomas and airways of SIV/Mtb co-infected animals. Taken together, these data show that pre-existing SIV alters T cell phenotypes and reduces TNF responses early in Mtb infection.

Influenza D viruses (IDV) belong to a new genus in the family Orthomyxoviridae. IDV is the aetiologic agent of acute, mild respiratory disease in ungulate species with agricultural importance (cattle, pigs, sheep, goats, camels, etc.). Despite the initial isolate being of porcine origin, serological data suggest cattle to be the primary host of IDV. The study aims were twofold: elucidating species-specific replication kinetics of IDV in bovine and porcine hosts and defining the interspecies potential with two different IDV strains. Three calves and three pigs were intranasally inoculated with the prototypic strain D/swine/Oklahoma/1334/2017 or a genetically distinct cattle isolate, D/bovine/Texas/72/2017. Two days following infection, three naive pigs and three naive calves were co-housed with inoculated calves and pigs, respectively. The species of IDV origin had no effect on virus replication kinetics in the upper respiratory tract of inoculated calves and pigs; similar shedding profiles were observed for each species and virus. However, interspecies transmission was found to be associated with virus origin species; D/bovine/Texas/72/2017 and D/swine/Oklahoma/1334/2017 were directly transmitted only to contact calves or pigs, respectively. Even so, transmission efficiency was higher for calves compared to pigs. Together, these data show that cattle and pigs are permissive for IDV replication, but IDV transmission may be species dependent. Host-specific mutations likely influenced transmission efficiencies between agriculturally important mammalian species.

The zoonotic emerging Rift Valley fever virus (RVFV) causes sporadic disease in livestock and humans throughout Africa and the Saudi Arabian peninsula. Infection of people with RVFV can occur through mosquito bite or mucosal exposure during butchering or milking of infected livestock. Disease typically presents as a self-limiting fever; however, in rare cases, hepatitis, encephalitis and ocular disease may occur. Recent studies have illuminated the neuropathogenic mechanisms of RVFV in a rat aerosol infection model. Neurological disease in rats is characterized by breakdown of the blood–brain barrier late in infection, infiltration of leukocytes to the central nervous system (CNS) and massive viral replication in the brain. However, the route of RVFV entry into the CNS after inhalational exposure remains unknown. Here, we visualized the entire nasal olfactory route from snout to brain after RVFV infection using RNA in situ hybridization and immunofluorescence microscopy. We found widespread RVFV-infected cells within the olfactory epithelium, across the cribriform plate, and in the glomerular region of the olfactory bulb within 2 days of infection. These results indicate that the olfactory tract is a major route of infection of the brain after inhalational exposure. A better understanding of potential neuroinvasion pathways can support the design of more effective therapeutic regiments for the treatment of neurological disease caused by RVFV.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), emerged at the end of 2019 and by mid-June 2020 the virus had spread to at least 215 countries, caused more than 8 000 000 confirmed infections and over 450 000 deaths, and overwhelmed healthcare systems worldwide. Like severe acute respiratory syndrome coronavirus (SARS-CoV), which emerged in 2002 and caused a similar disease, SARS-CoV-2 is a betacoronavirus. Both viruses use human angiotensin-converting enzyme 2 (hACE2) as a receptor to enter cells. However, the SARS-CoV-2 spike (S) glycoprotein has a novel insertion that generates a putative furin cleavage signal and this has been postulated to expand the host range. Two low-passage (P) strains of SARS-CoV-2 (Wash1 : P4 and Munich : P1) were cultured twice in Vero E6 cells and characterized virologically. Sanger and MinION sequencing demonstrated significant deletions in the furin cleavage signal of Wash1 : P6 and minor variants in the Munich : P3 strain. Cleavage of the S glycoprotein in SARS-CoV-2-infected Vero E6 cell lysates was inefficient even when an intact furin cleavage signal was present. Indirect immunofluorescence demonstrated that the S glycoprotein reached the cell surface. Since the S protein is a major antigenic target for the development of neutralizing antibodies, we investigated the development of neutralizing antibody titres in serial serum samples obtained from COVID-19 human patients. These were comparable regardless of the presence of an intact or deleted furin cleavage signal. These studies illustrate the need to characterize virus stocks meticulously prior to performing either in vitro or in vivo pathogenesis studies.

Immunomodulatory drugs could contribute to a functional cure for Human Immunodeficiency Virus (HIV). Interleukin-15 (IL-15) promotes expansion and activation of CD8+ T cell and natural killer (NK) cell populations. In one study, an IL-15 superagonist, N-803, suppressed Simian Immunodeficiency Virus (SIV) in non-human primates (NHPs) who had received prior SIV vaccination. However, viral suppression attenuated with continued N-803 treatment, partially returning after long treatment interruption. While there is evidence of concurrent drug tolerance, immune regulation, and viral escape, the relative contributions of these mechanisms to the observed viral dynamics have not been quantified. Here, we utilize mathematical models of N-803 treatment in SIV-infected macaques to estimate contributions of these three key mechanisms to treatment outcomes: 1) drug tolerance, 2) immune regulation, and 3) viral escape. We calibrated our model to viral and lymphocyte responses from the above-mentioned NHP study. Our models track CD8+ T cell and NK cell populations with N-803-dependent proliferation and activation, as well as viral dynamics in response to these immune cell populations. We compared mathematical models with different combinations of the three key mechanisms based on Akaike Information Criterion and important qualitative features of the NHP data. Two minimal models were capable of reproducing the observed SIV response to N-803. In both models, immune regulation strongly reduced cytotoxic cell activation to enable viral rebound. Either long-term drug tolerance or viral escape (or some combination thereof) could account for changes to viral dynamics across long breaks in N-803 treatment. Theoretical explorations with the models showed that less-frequent N-803 dosing and concurrent immune regulation blockade (e.g. PD-L1 inhibition) may improve N-803 efficacy. However, N-803 may need to be combined with other immune therapies to countermand viral escape from the CD8+ T cell response. Our mechanistic model will inform such therapy design and guide future studies., Author summary Immune therapy may be a critical component in the functional cure for Human Immunodeficiency Virus (HIV). N-803 is an immunotherapeutic drug that activates antigen-specific CD8+ T cells of the immune system. These CD8+ T cells eliminate HIV-infected cells in order to limit the spread of infection in the body. In one study, N-803 reduced plasma viremia in macaques that were infected with Simian Immunodeficiency Virus, an analog of HIV. Here, we used mathematical models to analyze the data from this study to better understand the effects of N-803 therapy on the immune system. Our models indicated that inhibitory signals may be reversing the stimulatory effect of N-803. Results also suggested the possibilities that tolerance to N-803 could build up within the CD8+ T cells themselves and that the treatment may be selecting for virus strains that are not targeted by CD8+ T cells. Our models predict that N-803 therapy may be made more effective if the time between doses is increased or if inhibitory signals are blocked by an additional drug. Also, N-803 may need to be combined with other immune therapies to target virus that would otherwise evade CD8+ T cells.

Influenza A viruses (IAV) sporadically transmit from swine to humans, typically associated with agricultural fairs in the United States. A human seasonal H3 virus from the 2010-2011 IAV season was introduced into the U.S. swine population and termed H3.2010.1 to differentiate it from the previous swine H3 virus. This H3N2 lineage became widespread in the U.S. commercial swine population, subsequently spilling over into exhibition swine, and caused a majority of H3N2 variant (H3N2v) cases in humans in 2016 and 2017. A cluster of human H3N2v cases were reported at an agricultural fair in 2017 in Ohio, where 2010.1 H3N2 IAV was concurrently detected in exhibition swine. Genomic analysis showed that the swine and human isolates were nearly identical. In this study, we evaluated the propensity of a 2010.1 H3N2 IAV (A/swine/Ohio/A01354299/2017 [sw/OH/2017]) isolated from a pig in the agricultural fair outbreak to replicate in ferrets and transmit from swine to ferret. sw/OH/2017 displayed robust replication in the ferret respiratory tract, causing slight fever and moderate weight loss. Further, sw/OH/2017 was capable of efficient respiratory droplet transmission from infected pigs to contact ferrets. These findings establish a model for evaluating the propensity of swine IAV to transmit from pig to ferret as a measure of risk to the human population. The identification of higher-risk swine strains can then be targeted for control measures to limit the dissemination at human-swine interfaces to reduce the risk of zoonotic infections and to inform pandemic planning. IMPORTANCE A recently emerged lineage of human-like H3N2 (H3.2010.1) influenza A virus (IAV) from swine has been frequently detected in commercial and exhibition swine in recent years and has been associated with H3N2 variant cases in humans from 2016 and 2017. To demonstrate a model for characterizing the potential for zoonotic transmission associated with swine IAV, we performed an in vivo study of transmission between pigs infected with an H3.2010.1 H3N2 IAV and aerosol contact ferrets. The efficient interspecies transmission demonstrated for the H3.2010.1 IAV in swine emphasizes the need for further characterization of viruses circulating at the swine-human interface for transmission potential prior to human spillover and the development and implementation of more robust vaccines and control strategies to mitigate human exposure to higher-risk swine strains.

The observed COVID-19 case-fatality rate varies widely across countries. This study determined how much of the variation in case-fatality rate was the result of country-specific age-related factors (that is, age distribution of the cases and population age distribution). The information is critical for making meaningful comparisons of COVID-19 case-fatality rates across countries., Visual Abstract. Age Distributions of Cases and COVID-19 Case Fatality Across Countries The observed COVID-19 case-fatality rate varies widely across countries. This study determined how much of the variation in case-fatality rate was the result of country-specific age-related factors (that is, age distribution of the cases and population age distribution). The information is critical for making meaningful comparisons of COVID-19 case-fatality rates across countries. Visual Abstract. Age Distributions of Cases and COVID-19 Case Fatality Across Countries The observed COVID-19 case-fatality rate varies widely across countries. This study determined how much of the variation in case-fatality rate was the result of country-specific age-related factors (that is, age distribution of the cases and population age distribution). The information is critical for making meaningful comparisons of COVID-19 case-fatality rates across countries., Background: There is wide variation in coronavirus disease 2019 (COVID-19) case-fatality rates (CFRs) across countries, leading to uncertainty about the true lethality of the disease. A large part of this variation may be due to the ages of individuals who are tested and identified. Objective: To measure the contribution of distortions from the age distributions of confirmed cases to CFRs within and across populations. Design: Cross-sectional demographic study using aggregate data on COVID-19 cases and deaths by age. Setting: Population-based data from China, France, Germany, Italy, the Netherlands, South Korea, Spain, Switzerland, and the United States. Participants: All individuals with confirmed COVID-19, as reported by each country as of 19 April 2020 (N = 1 223 261). Measurements: Age-specific COVID-19 CFRs and age-specific population shares by country. Results: The overall observed CFR varies widely, with the highest rates in Italy (9.3%) and the Netherlands (7.4%) and the lowest rates in South Korea (1.6%) and Germany (0.7%). Adjustment for the age distribution of cases explains 66% of the variation of across countries, with a resulting age-standardized median CFR of 1.9%. Among a larger sample of 95 countries, the observed variation in COVID-19 CFRs is 13 times larger than what would be expected on the basis of just differences in the age-composition of countries. Limitation: The age-adjusted rates assume that, conditional on age, COVID-19 mortality among diagnosed cases is the same as that among undiagnosed cases and that individuals of all ages are equally susceptible to severe acute respiratory syndrome coronavirus 2 infection. Conclusion: Selective testing and identifying of older cases considerably warps estimates of the lethality of COVID-19 within populations and comparisons across countries. Removing age distortions and focusing on differences in age-adjusted case fatality will be essential for accurately comparing countries' performance in caring for patients with COVID-19 and for monitoring the epidemic over time. Primary Funding Source: Alexander von Humboldt Foundation.

Infections caused by Gram-negative bacteria are difficult to fight because these pathogens exclude or expel many clinical antibiotics and host defense molecules. However, mammals have evolved a substantial immune arsenal that weakens pathogen defenses, suggesting the feasibility of developing therapies that work in concert with innate immunity to kill Gram-negative bacteria. Using chemical genetics, we recently identified a small molecule, JD1, that kills Salmonella enterica serovar Typhimurium (S. Typhimurium) residing within macrophages. JD1 is not antibacterial in standard microbiological media, but rapidly inhibits growth and curtails bacterial survival under broth conditions that compromise the outer membrane or reduce efflux pump activity. Using a combination of cellular indicators and super resolution microscopy, we found that JD1 damaged bacterial cytoplasmic membranes by increasing fluidity, disrupting barrier function, and causing the formation of membrane distortions. We quantified macrophage cell membrane integrity and mitochondrial membrane potential and found that disruption of eukaryotic cell membranes required approximately 30-fold more JD1 than was needed to kill bacteria in macrophages. Moreover, JD1 preferentially damaged liposomes with compositions similar to E. coli inner membranes versus mammalian cell membranes. Cholesterol, a component of mammalian cell membranes, was protective in the presence of neutral lipids. In mice, intraperitoneal administration of JD1 reduced tissue colonization by S. Typhimurium. These observations indicate that during infection, JD1 gains access to and disrupts the cytoplasmic membrane of Gram-negative bacteria, and that neutral lipids and cholesterol protect mammalian membranes from JD1-mediated damage. Thus, it may be possible to develop therapeutics that exploit host innate immunity to gain access to Gram-negative bacteria and then preferentially damage the bacterial cell membrane over host membranes., Author summary Bacteria are increasingly becoming resistant to the antibiotics that are currently available. It has even been predicted that in the next thirty years there will be more deaths from antibiotic resistant infections than from cancer. We therefore need new antibiotics. To decrease the likelihood that bacteria will rapidly develop resistance to new antibacterials, researchers are seeking novel compounds that work differently than existing antibiotics. To find such a compound, we looked for chemicals that reduce the number of infectious bacteria within mammalian cells. We focused our efforts on Gram-negative bacteria because this class of pathogens is particularly difficult to treat with antibiotics. We present data showing that the compound JD1 disrupts bacterial cell membranes, a structure not targeted by current antibiotics for Gram-negative bacteria. JD1 also decreases bacterial colonization of infected mice. This is the first compound, to our knowledge, that preferentially targets the cell membranes of Gram-negative bacteria and reduces bacterial infection of animals.

The emergent coronavirus, designated severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), is a zoonotic pathogen that has demonstrated remarkable transmissibility in the human population and is the etiological agent of a current global pandemic called COVID-191. We measured the dynamic (short-term) aerosol efficiencies of SARS-CoV-2 and compared the efficiencies with two other emerging coronaviruses, SARS-CoV (emerged in 2002) and Middle Eastern respiratory syndrome CoV (MERS-CoV; emerged starting in 2012). We also quantified the long-term persistence of SARS-CoV-2 and its ability to maintain infectivity when suspended in aerosols for up to 16 hours.

Licensed vaccines or therapeutics are rarely available for pathogens with epidemic or pandemic potential. Developing interventions for specific pathogens and defining generalizable approaches for related pathogens is a global priority and inherent to the UN Sustainable Development Goals. Nipah virus (NiV) poses a significant epidemic threat, and zoonotic transmission from bats-to-humans with high fatality rates occurs almost annually. Human-to-human transmission of NiV has been documented in recent outbreaks leading public health officials and government agencies to declare an urgent need for effective vaccines and therapeutics. Here, we evaluate NiV vaccine antigen design options including the fusion glycoprotein (F) and the major attachment glycoprotein (G). A stabilized prefusion F (pre-F), multimeric G constructs, and chimeric proteins containing both pre-F and G were developed as protein subunit candidate vaccines. The proteins were evaluated for antigenicity and structural integrity using kinetic binding assays, electron microscopy, and other biophysical properties. Immunogenicity of the vaccine antigens was evaluated in mice. The stabilized pre-F trimer and hexameric G immunogens both induced serum neutralizing activity in mice, while the post-F trimer immunogen did not elicit neutralizing activity. The pre-F trimer covalently linked to three G monomers (pre-F/G) induced potent neutralizing antibody activity, elicited responses to the greatest diversity of antigenic sites, and is the lead candidate for clinical development. The specific stabilizing mutations and immunogen designs utilized for NiV were successfully applied to other henipaviruses, supporting the concept of identifying generalizable solutions for prototype pathogens as an approach to pandemic preparedness.

The poxvirus, myxoma virus (MYXV) has shown efficacy as an oncolytic virus (OV) in some cancer models. However, MYXV replication within murine cancer models and spontaneous canine sarcomas is short-lived. In mice, successful treatment of tumors requires frequent injections with MYXV. We hypothesize that treatment of cancer with a recombinant MYXV that promotes apoptosis could improve the efficacy of MYXV. The orfC gene of walleye dermal sarcoma virus (WDSV), which induces apoptosis, was recombined into the MYXV genome (MYXVorfC). A marked increase in apoptosis was observed in cells infected with MYXVorfC. To ensure that expression of WDSV orfC by MYXV does not potentiate the pathogenesis of MYXV, we evaluated the effects of MYXVorfC inoculation in the only known host of MYXV, New Zealand white rabbits. Virus dissemination in rabbit tissues was similar for MYXVorfC and MYXV. Virus titers recovered from tissues were lower in MYXVorfC-infected rabbits as compared to MYXV-infected rabbits. Importantly, rabbits infected with MYXVorfC had a delayed onset of clinical signs and a longer median survival time than rabbits infected with MYXV. This study indicates that MYXVorfC is attenuated and suggests that MYXVorfC will be safe to use as an OV therapy in future studies.

Swine are influenza virus reservoirs that have caused outbreaks and pandemics. Genomic characterization of these viruses enables pandemic risk assessment and vaccine comparisons, though this typically occurs after a novel swine virus jumps into humans. The greatest risk occurs where large groups of swine and humans comingle. At a large swine exhibition, we used Nanopore sequencing and on-site analytics to interpret 13 swine influenza virus genomes and identified an influenza virus cluster that was genetically highly varied to currently available vaccines. As part of the National Strategy for Pandemic Preparedness exercises, the sequences were emailed to colleagues at the CDC who initiated the development of a synthetically derived vaccine designed to match the viruses at the exhibition. Subsequently, this virus caused 14 infections in humans and was the dominant U.S. variant virus in 2018., While working overnight at a swine exhibition, we identified an influenza A virus (IAV) outbreak in swine, Nanopore sequenced 13 IAV genomes from samples we collected, and predicted in real time that these viruses posed a novel risk to humans due to genetic mismatches between the viruses and current prepandemic candidate vaccine viruses (CVVs). We developed and used a portable IAV sequencing and analysis platform called Mia (Mobile Influenza Analysis) to complete and characterize full-length consensus genomes approximately 18 h after unpacking the mobile lab. Exhibition swine are a known source for zoonotic transmission of IAV to humans and pose a potential pandemic risk. Genomic analyses of IAV in swine are critical to understanding this risk, the types of viruses circulating in swine, and whether current vaccines developed for use in humans would be predicted to provide immune protection. Nanopore sequencing technology has enabled genome sequencing in the field at the source of viral outbreaks or at the bedside or pen-side of infected humans and animals. The acquired data, however, have not yet demonstrated real-time, actionable public health responses. The Mia system rapidly identified three genetically distinct swine IAV lineages from three subtypes, A(H1N1), A(H3N2), and A(H1N2). Analysis of the hemagglutinin (HA) sequences of the A(H1N2) viruses identified >30 amino acid differences between the HA1 of these viruses and the most closely related CVV. As an exercise in pandemic preparedness, all sequences were emailed to CDC collaborators who initiated the development of a synthetically derived CVV. IMPORTANCE Swine are influenza virus reservoirs that have caused outbreaks and pandemics. Genomic characterization of these viruses enables pandemic risk assessment and vaccine comparisons, though this typically occurs after a novel swine virus jumps into humans. The greatest risk occurs where large groups of swine and humans comingle. At a large swine exhibition, we used Nanopore sequencing and on-site analytics to interpret 13 swine influenza virus genomes and identified an influenza virus cluster that was genetically highly varied to currently available vaccines. As part of the National Strategy for Pandemic Preparedness exercises, the sequences were emailed to colleagues at the CDC who initiated the development of a synthetically derived vaccine designed to match the viruses at the exhibition. Subsequently, this virus caused 14 infections in humans and was the dominant U.S. variant virus in 2018.

Mucosal-associated invariant T (MAIT) cells can recognize and respond to some bacterially infected cells. Several in vitro and in vivo models of Mycobacterium tuberculosis (Mtb) infection suggest that MAIT cells can contribute to control of Mtb, but these studies are often cross-sectional and use peripheral blood cells. Whether MAIT cells are recruited to Mtb-affected granulomas and lymph nodes (LNs) during early Mtb infection and what purpose they might serve there is less well understood. Furthermore, whether HIV/SIV infection impairs MAIT cell frequency or function at the sites of Mtb replication has not been determined. Using Mauritian cynomolgus macaques (MCM), we phenotyped MAIT cells in the peripheral blood and bronchoalveolar lavage (BAL) before and during infection with SIVmac239. To test the hypothesis that SIV co-infection impairs MAIT cell frequency and function within granulomas, SIV+ and -naïve MCM were infected with a low dose of Mtb Erdman, and necropsied at 6 weeks post Mtb-challenge. MAIT cell frequency and function were examined within the peripheral blood, BAL, and Mtb-affected lymph nodes (LN) and granulomas. MAIT cells did not express markers indicative of T cell activation in response to Mtb in vivo within granulomas in animals infected with Mtb alone. SIV and Mtb co-infection led to increased expression of the activation/exhaustion markers PD-1 and TIGIT, and decreased ability to secrete TNFα when compared to SIV-naïve MCM. Our study provides evidence that SIV infection does not prohibit the recruitment of MAIT cells to sites of Mtb infection, but does functionally impair those MAIT cells. Their impaired function could have impacts, either direct or indirect, on the long-term containment of TB disease., Author summary MAIT cells are a population of immune cells that can directly detect and destroy some bacterially infected cells. Evidence suggests that MAIT cells may play a role in control of Mycobacterium tuberculosis (Mtb) infection, but few studies have examined MAIT cell activity within granulomas, which are the sites of Mtb replication. In addition, chronic HIV infection has been shown to impair the frequency and function of MAIT cells in humans, but these studies focus on peripheral blood and not the sites of Mtb infection. Here, we used a macaque model of SIV and Mtb co-infection to determine whether SIV, as a model for HIV, could dysregulate MAIT cells in tissues where Mtb replication is occurring. SIV co-infection did not affect the absolute numbers of MAIT cells present within granulomas but did impair the ability of the MAIT cells to respond to mycobacteria both in vivo and in vitro. Overall, our study provides evidence that SIV infection alters MAIT cells phenotypically, and impairs MAIT cell function. What effect this might have on antimycobacterial immunity is an avenue for future exploration.

The neuraminidase (NA) of influenza A viruses (IAV) is a structurally and antigenically important envelope glycoprotein. There are eleven known subtypes of NA of which two, N1 and N2, circulate in swine. The sialidase activity of NA is required for the release of nascent virus particles from infected cell membranes and inhibition of NA enzymatic activity can significantly reduce virus titers and duration of infection. Efforts to improve IAV vaccine technology in humans have focused on the generation of neuraminidase inhibiting (NAI) antibodies and should be considered in swine as well. The enzyme-linked lectin assay (ELLA) conducted in 96-well plates has enabled high-throughput analysis of serum samples for NAI antibody titers. Through the use of reverse genetics, custom antigen panels and antisera can be generated to encompass the antigenically diverse population of NA that circulate in swine. The ELLA is a robust method to assess NAI antibody titers and characterize the antigenic difference between NA antigens.

Chickens infected with avian influenza virus (AIV) transmit the virus via respiratory and cloacal shedding. While previous mathematical models have shown that the innate immune response is necessary for the early suppression of virus production in infected respiratory cells, the different pathways by which the innate immune response can affect cloacal viral shedding have not been studied in chickens. The present study aims to evaluate the sensitivity of H9N2 low pathogenic AIV shedding in chicken gastrointestinal cells to different type-I interferon (IFN) response pathways, and to determine the impact of a cellular eclipse phase (latent period) on the time to peak virus shedding using a mathematical model describing within host viral kinetics. Our model results demonstrate that a mechanistic model that incorporates 1) the intracellular antiviral effects of type-I IFN on virus production, 2) destruction of infected cells by type-I IFN activated Natural Killer cells, and 3) an eclipse phase is most consistent with experimental cloacal virus shedding data. These results provide a potential mechanistic explanation for the delay to peak cloacal virus shedding observed in experimental studies conducted in chickens, as well as an improved understanding of the primary type-I IFN pathways involved in the control of cloacal virus shedding, which may lead to the development of more targeted vaccine candidates.

Mycoplasma cynos is recognized as an emerging causative pathogen of canine infectious respiratory disease (CIRD) worldwide. We developed a new open-source real-time PCR (rtPCR) assay for M. cynos that performs well under standard rtPCR conditions. Primers and probes were designed to target the M. cynos tuf gene. Reaction efficiencies for the M. cynos tuf gene assay on 2 platforms were based on amplification of standard curves spanning 8 orders of magnitude: ABI 7500 platform, 94.3–97.9% ( r2 ≥ 0.9935); QuantStudio OpenArray platform, 119.1–122.5% ( r2 = 0.9784). The assay performed very well over a range of template input, from 109 copies to the lower limit of quantification at 4 copies of the M. cynos genome on the ABI 7500 platform. Diagnostic performance was estimated by comparison with an in-house legacy assay on clinical specimens as well as testing isolates that were characterized previously by intergenic spacer region (ISR) sequencing. Exclusivity was established by testing 12 other Mycoplasma species. To substantiate the high specificity of the M. cynos tuf gene assay, sequence confirmation was performed on ISR PCR amplicons obtained from clinical specimens. One ISR amplicon sequence revealed M. mucosicanis rather than M. cynos. The complete protocol of the newly developed M. cynos tuf assay is provided to facilitate assay harmonization.

Eastern (EEEV) and Venezuelan (VEEV) equine encephalitis viruses (EEVs) are related, (+) ssRNA arboviruses that can cause severe, sometimes fatal, encephalitis in humans. EEVs are highly infectious when aerosolized, raising concerns for potential use as biological weapons. No licensed medical countermeasures exist, given the severity/rarity of natural EEV infections, efficacy studies require animal models. Cynomolgus macaques exposed to EEV aerosols develop fever, encephalitis, and other clinical signs similar to humans. Fever is nonspecific for encephalitis in macaques. Electrocardiography (ECG) metrics may predict onset, severity, or outcome of EEV-attributable disease. Macaques were implanted with thermometry/ECG radiotransmitters and exposed to aerosolized EEV. Data was collected continuously, and repeated-measures ANOVA and frequency-spectrum analyses identified differences between courses of illness and between pre-exposure and post-exposure states. EEEV-infected macaques manifested widened QRS-intervals in severely ill subjects post-exposure. Moreover, QT-intervals and RR-intervals decreased during the febrile period. VEEV-infected macaques suffered decreased QT-intervals and RR-intervals with fever onset. Frequency-spectrum analyses revealed differences in the fundamental frequencies of multiple metrics in the post-exposure and febrile periods compared to baseline and confirmed circadian dysfunction. Heart rate variability (HRV) analyses revealed diminished variability post-exposure. These analyses support using ECG data alongside fever and clinical laboratory findings for evaluating medical countermeasure efficacy.

Several lineages of influenza A viruses (IAV) currently circulate in North American pigs. Genetic diversity is further increased by transmission of IAV between swine and humans and subsequent evolution. Here, we characterized the genetic and antigenic evolution of contemporary swine H1N1 and H1N2 viruses representing clusters H1-α (1A.1), H1-β (1A.2), H1pdm (1A.3.3.2), H1-γ (1A.3.3.3), H1-δ1 (1B.2.2), and H1-δ2 (1B.2.1) currently circulating in pigs in the United States. The δ1-viruses diversified into two new genetic clades, H1-δ1a (1B.2.2.1) and H1-δ1b (1B.2.2.2), which were also antigenically distinct from the earlier H1-δ1-viruses. Further characterization revealed that a few key amino acid changes were associated with antigenic divergence in these groups. The continued genetic and antigenic evolution of contemporary H1 viruses might lead to loss of vaccine cross-protection that could lead to significant economic impact to the swine industry, and represents a challenge to public health initiatives that attempt to minimize swine-to-human IAV transmission.

Papillomaviridae is a diverse family of circular, double-stranded DNA (dsDNA) viruses that infect a broad range of mammalian, avian and fish hosts. While papillomaviruses have been characterized most extensively in humans, the study of non-human papillomaviruses has contributed greatly to our understanding of their pathogenicity and evolution. Using high-throughput sequencing approaches, we identified 7 novel papillomaviruses from vaginal swabs collected from 81 adult female Weddell seals (Leptonychotes weddellii) in the Ross Sea of Antarctica between 2014-2017. These seven papillomavirus genomes were amplified from seven individual seals, and six of the seven genomes represented novel species with distinct evolutionary lineages. This highlights the diversity of papillomaviruses among the relatively small number of Weddell seal samples tested. Viruses associated with large vertebrates are poorly studied in Antarctica, and this study adds information about papillomaviruses associated with Weddell seals and contributes to our understanding of the evolutionary history of papillomaviruses.

Influenza A virus (IAV) in swine constitutes a major economic burden for producers as well as a potential threat to public health. Whole inactivated virus vaccines (WIV) are the predominant countermeasure employed to control IAV in swine herds in the United States despite the superior protection, and diminished adverse effects, induced by live attenuated influenza vaccines (LAIV). A major hurdle for the development of LAIV exists in achieving the proper level of attenuation while maintaining immunogenicity. Using Synthetic Attenuated Virus Engineering (SAVE) to introduce codon-pair bias de-optimization (CPBD) into the hemagglutinin (HA) and neuraminidase (NA) gene segments of pandemic H1N1 IAV, a novel LAIV was produced and evaluated for attenuation, immunogenicity, and efficacy in pigs. The CPBD LAIV induced inappreciable pathology following intranasal administration yet induced robust serum and mucosal antibody titers. CPBD LAIV vaccinated pigs challenged with wild-type virus showed protection from disease and virus detection, highlighted by the absence of detectable virus titers in the nasal passages and lungs. These results demonstrate the efficacy of a LAIV designed by SAVE codon de-optimization in pigs, providing support for the continued development of CPBD LAIV for use in swine.

Introduction Zika virus (ZIKV) is primarily transmitted byAedes aegypti and Aedes albopictus mosquitoes between humans and non-human primates. Climate change may enhance virus reproduction in Aedes spp. mosquito populations, resulting in intensified ZIKV outbreaks. The study objective was to explore how an outbreak similar to the 2016 ZIKV outbreak in Brazil might unfold with projected climate change. Methods A compartmental infectious disease model that included compartments for humans and mosquitoes was developed to fit the 2016 ZIKV outbreak data from Brazil using least squares optimization. To explore the impact of climate change, published polynomial relationships between temperature and temperature-sensitive mosquito population and virus transmission parameters (mosquito mortality, development rate, and ZIKV extrinsic incubation period) were used. Projections for future outbreaks were obtained by simulating transmission with effects of projected average monthly temperatures on temperature-sensitive model parameters at each of three future time periods: 2011–2040, 2041–2070, and 2071–2100. The projected future climate was obtained from an ensemble of regional climate models (RCMs) obtained from the Co-Ordinated Regional Downscaling Experiment (CORDEX) that used Representative Concentration Pathways (RCP) with two radiative forcing values, RCP4.5 and RCP8.5. A sensitivity analysis was performed to explore the impact of temperature-dependent parameters on the model outcomes. Results Climate change scenarios impacted the model outcomes, including the peak clinical case incidence, cumulative clinical case incidence, time to peak incidence, and the duration of the ZIKV outbreak. Comparing 2070–2100 to 2016, using RCP4.5, the peak incidence was 22,030 compared to 10,473; the time to epidemic peak was 12 compared to 9 weeks, and the outbreak duration was 52 compared to 41 weeks. Comparing 2070–2100 to 2016, using RCP8.5, the peak incidence was 21,786 compared to 10,473; the time to epidemic peak was 11 compared to 9 weeks, and the outbreak duration was 50 compared to 41weeks. The increases are due to optimal climate conditions for mosquitoes, with the mean temperature reaching 28 °C in the warmest months. Under a high emission scenario (RCP8.5), mean temperatures extend above optimal for mosquito survival in the warmest months. Conclusion Outbreaks of ZIKV in locations similar to Brazil are expected to be more intense with a warming climate. As climate change impacts are becoming increasingly apparent on human health, it is important to quantify the effect and use this knowledge to inform decisions on prevention and control strategies.

A canine influenza A(H3N2) virus emerged in the United States in February-March 2015, causing respiratory disease in dogs. The virus had previously been circulating among dogs in Asia, where it originated through the transfer of an avian-origin influenza virus around 2005 and continues to circulate. Sequence analysis suggests the US outbreak was initiated by a single introduction, in Chicago, of an H3N2 canine influenza virus circulating among dogs in South Korea in 2015. Despite local control measures, the virus has continued circulating among dogs in and around Chicago and has spread to several other areas of the country, particularly Georgia and North Carolina, although these secondary outbreaks appear to have ended within a few months. Some genetic variation has accumulated among the US viruses, with the appearance of regional-temporal lineages. The potential for interspecies transmission and zoonotic events involving this newly emerged influenza A virus is currently unknown.

Rift Valley Fever (RVF) is an emerging arboviral disease of livestock and humans. Although the disease is caused by a mosquito-borne virus, humans are infected through contact with, or inhalation of, virus-laden particles from contaminated animal carcasses. Some individuals infected with RVF virus (RVFV) develop meningoencephalitis, resulting in morbidity and mortality. Little is known about the pathogenic mechanisms that lead to neurologic sequelae, and thus, animal models that represent human disease are needed. African green monkeys (AGM) exposed to aerosols containing RVFV develop a reproducibly lethal neurological disease that resembles human illness. To understand the disease process and identify biomarkers of lethality, two groups of 5 AGM were infected by inhalation with either a lethal or a sublethal dose of RVFV. Divergence between lethal and sublethal infections occurred as early as 2 days postinfection (dpi), at which point CD8 + T cells from lethally infected AGM expressed activated caspase-3 and simultaneously failed to increase levels of major histocompatibility complex (MHC) class II molecules, in contrast to surviving animals. At 4 dpi, lethally infected animals failed to demonstrate proliferation of total CD4 + and CD8 + T cells, in contrast to survivors. These marked changes in peripheral blood cells occur much earlier than more-established indicators of severe RVF disease, such as granulocytosis and fever. In addition, an early proinflammatory (gamma interferon [IFN-γ], interleukin 6 [IL-6], IL-8, monocyte chemoattractant protein 1 [MCP-1]) and antiviral (IFN-α) response was seen in survivors, while very late cytokine expression was found in animals with lethal infections. By characterizing immunological markers of lethal disease, this study furthers our understanding of RVF pathogenesis and will allow the testing of therapeutics and vaccines in the AGM model. IMPORTANCE Rift Valley Fever (RVF) is an important emerging viral disease for which we lack both an effective human vaccine and treatment. Encephalitis and neurological disease resulting from RVF lead to death or significant long-term disability for infected people. African green monkeys (AGM) develop lethal neurological disease when infected with RVF virus by inhalation. Here we report the similarities in disease course between infected AGM and humans. For the first time, we examine the peripheral immune response during the course of infection in AGM and show that there are very early differences in the immune response between animals that survive infection and those that succumb. We conclude that AGM are a novel and suitable monkey model for studying the neuropathogenesis of RVF and for testing vaccines and therapeutics against this emerging viral pathogen.

There were an estimated 10 million new cases of tuberculosis (TB) disease in 2019. While over 90% of individuals successfully control Mycobacterium tuberculosis (Mtb) infection, which causes TB disease, HIV co-infection often leads to active TB disease. Despite the co-endemic nature of HIV and TB, knowledge of the immune mechanisms contributing to the loss of control of Mtb replication during HIV infection is lacking. Mucosal-associated invariant T (MAIT) cells are innate-like T cells that target and destroy bacterially-infected cells and may contribute to the control of Mtb infection. Studies examining MAIT cells in human Mtb infection are commonly performed using peripheral blood samples. However, because Mtb infection occurs primarily in lung tissue and lung-associated lymph nodes, these studies may not be fully translatable to the tissues. Additionally, studies longitudinally examining MAIT cell dynamics during HIV/Mtb co-infection are rare, and lung and lymph node tissue samples from HIV+ patients are typically unavailable. Nonhuman primates (NHP) provide a model system to characterize MAIT cell activity during Mtb infection, both in Simian Immunodeficiency Virus (SIV)-infected and SIV-naïve animals. Using NHPs allows for a more comprehensive understanding of tissue-based MAIT cell dynamics during infection with both pathogens. NHP SIV and Mtb infection is similar to human HIV and Mtb infection, and MAIT cells are phenotypically similar in humans and NHPs. Here, we discuss current knowledge surrounding MAIT cells in SIV and Mtb infection, how SIV infection impairs MAIT cell function during Mtb co-infection, and knowledge gaps to address.

The porcine reproductive and respiratory syndrome virus (PRRSV) is an enveloped RNA virus, with high mutation rates and genetic variability ; which is evident by the large number of discrete strains that co-circulate in swine populations. Veterinary practitioners frequently identify certain discrete PRRSV strains as having a higher clinical impact on production. However, with exception of a few strains, production impact is not well characterized for the majority of PRRSV variants. Predictive analytics, coupled with routine diagnostic sequencing of PRRSV, provide opportunities to study the clinical impact of discrete PRRSV strains on production. Thus, the primary objective of this research was to evaluate clinical impact of discrete PRRSV clades observed in Ontario sow farms. PRRS viruses were classified into discrete clades using Bayesian analysis of the nucleotide sequences of the ORF-5 region of the genome. Production data were gathered through veterinary clinics from herds participating in the ongoing PRRSV surveillance system. Data about pre-weaning mortality, sow mortality, and abortion rates were measured up to 8 weeks post initial PRRSV outbreak. Through conventional regression analysis, results support that clinical impact of the viruses varied among clades over time for abortion rate (p = 0.05) and pre-weaning mortality (p < 0.01). Using predictive modelling approaches based on grouped K-fold cross-validation, it was identified that PRRSV clade designations and other measured factors showed low predictive performance for abortion (R2 = 0.07), pre-weaning mortality (R2 = 0.09), and sow mortality (R2 = 0.04). Clade designation consistently showed moderate importance for abortion and pre-weaning mortality, with clade 2 viruses being identified, on average, as having higher impact. These results demonstrate that the prediction of clinical impact, through production parameters, based on phylogenetic classification of PRRS viruses is possible. However, very high impact outbreaks were difficult to predict across production parameters. More surveillance- derived data are required to continue to improve predictive performance of the models.

Background In the fall of 2014, highly pathogenic avian influenza (HPAI) subtype H5N8 clade 2.3.4.4 was introduced into North America by migrating waterfowl from Asia where, through reassortment, novel HPAI H5N2 and H5N1 viruses emerged. Objectives Assess the susceptibility of pigs to HPAI H5N1, H5N2, and H5N8 clade 2.3.3.3 from North America. Methods Pigs and trachea explants were inoculated with a representative panel of H5NX clade 2.3.4.4 HPAI viruses from North America. Nasal swabs, BALF, and sera were collected to assess replication and transmission in challenged and direct contact pigs by RRT-PCR, virus isolation, hemagglutination inhibition, and ELISA. Results Limited virus replication was restricted to the lower respiratory tract of challenged pigs, though absent in the nasal passages and trachea cultures, as determined by RRT-PCR in all samples. Seroconversion of inoculated pigs was detected by NP ELISA but was not reliably detected by antigen-specific hemagglutination inhibition. Boost with adjuvanted virus was required for the production of neutralizing antibodies to assess cross-reactivity between wild-type avian strains. All RRT-PCR and serology tests were negative for contact animals indicating a failure of transmission from primary inoculated pigs. Conclusions H5NX clade 2.3.4.4 strains can replicate in the lower respiratory tract of swine upon high titer inoculation, though appear to be incapable of replication in swine nasal epithelium in vivo or ex vivo in trachea explants in culture. Infected pigs did not produce high levels of serum antibodies following infection. Collectively, our data show HPAI H5NX clade 2.3.4.4 viruses to be poorly adapted for replication and transmission in swine.