Publications

Using multiple viral systems, and performing silencing approaches, overexpression approaches, and experiments in knockout cells, we report, for the first time, that interferon (IFN)-induced protein 44 (IFI44) positively affects virus production and negatively modulates innate immune responses induced after viral infections. Moreover, IFI44 is able to rescue poly(I·C)- and IFN-mediated inhibition of virus growth. Furthermore, we report a novel interaction of IFI44 with the cellular factor FK506-binding protein 5 (FKBP5), which binds to cellular kinases such as the inhibitor of nuclear factor kappa B (IκB) kinases (IKKα, IKKβ, and IKKε). Importantly, in the presence of FKBP5, IFI44 decreases the ability of IKKβ to phosphorylate IκBα and the ability of IKKε to phosphorylate interferon regulatory factor 3 (IRF-3), providing a novel mechanism for the function of IFI44 in negatively modulating IFN responses. Remarkably, these new IFI44 functions may have implications for diseases associated with excessive immune signaling and for controlling virus infections mediated by IFN responses.IMPORTANCE Innate immune responses mediated by IFN and inflammatory cytokines are critical for controlling virus replication. Nevertheless, exacerbated innate immune responses could be detrimental for the host and feedback mechanisms are needed to maintain the cellular homeostasis. In this work, we describe a completely novel function for IFI44 in negatively modulating the innate immune responses induced after viral infections. We show that decreasing IFI44 expression by using small interfering RNAs (siRNAs) or by generating knockout (KO) cells impairs virus production and increases the levels of IFN responses. Moreover, we report a novel interaction of IFI44 with the cellular protein FKBP5, which in turn interacts with kinases essential for type I and III IFN induction and signaling, such as the inhibitor of nuclear factor kappa B (IκB) kinases IKKα, IKKβ, and IKKε. Our data indicate that binding of IFI44 to FKBP5 decreased the phosphorylation of IRF-3 and IκBα mediated by IKKε and IKKβ, respectively, providing a likely explanation for the function of IFI44 in negatively modulating IFN responses. These results provide new insights into the induction of innate immune responses and suggest that IFI44 is a new potential antiviral target for reducing virus replication.

We describe a novel function for the interferon (IFN)-induced protein 44-like (IFI44L) gene in negatively modulating innate immune responses induced after virus infections. Furthermore, we show that decreasing IFI44L expression impairs virus production and that IFI44L expression negatively modulates the antiviral state induced by an analog of double-stranded RNA (dsRNA) or by IFN treatment. The mechanism likely involves the interaction of IFI44L with cellular FK506-binding protein 5 (FKBP5), which in turn interacts with kinases essential for type I and III IFN responses, such as inhibitor of nuclear factor kappa B (IκB) kinase alpha (IKKα), IKKβ, and IKKε. Consequently, binding of IFI44L to FKBP5 decreased interferon regulatory factor 3 (IRF-3)-mediated and nuclear factor kappa-B (NF-κB) inhibitor (IκBα)-mediated phosphorylation by IKKε and IKKβ, respectively. According to these results, IFI44L is a good target for treatment of diseases associated with excessive IFN levels and/or proinflammatory responses and for reduction of viral replication.IMPORTANCE Excessive innate immune responses can be deleterious for the host, and therefore, negative feedback is needed. Here, we describe a completely novel function for IFI44L in negatively modulating innate immune responses induced after virus infections. In addition, we show that decreasing IFI44L expression impairs virus production and that IFI44L expression negatively modulates the antiviral state induced by an analog of dsRNA or by IFN treatment. IFI44L binds to the cellular protein FKBP5, which in turn interacts with kinases essential for type I and III IFN induction and signaling, such as the kinases IKKα, IKKβ, and IKKε. IFI44L binding to FKBP5 decreased the phosphorylation of IRF-3 and IκBα mediated by IKKε and IKKβ, respectively, providing an explanation for the function of IFI44L in negatively modulating IFN responses. Therefore, IFI44L is a candidate target for reducing virus replication.

The critical step in the emergence of a new epidemic or pandemic viral pathogen occurs after it infects the initial spillover host and then is successfully transmitted onwards, causing an outbreak chain of transmission within that new host population. Crossing these choke points sets a pathogen on the pathway to epidemic emergence. While many viruses spill over to infect new or alternative hosts, only a few accomplish this transition-and the reasons for the success of those pathogens are still unclear. Here, we consider this issue related to the emergence of animal viruses, where factors involved likely include the ability to efficiently infect the new animal host, the demographic features of the initial population that favour onward transmission, the level of shedding and degree of susceptibility of individuals of that population, along with pathogen evolution favouring increased replication and more efficient transmission among the new host individuals. A related form of emergence involves mutations that increased spread or virulence of an already-known virus within its usual host. In all of these cases, emergence may be due to altered viral properties, changes in the size or structure of the host populations, ease of transport, climate change or, in the case of arboviruses, to the expansion of the arthropod vectors. Here, we focus on three examples of viruses that have gained efficient onward transmission after spillover: influenza A viruses that are respiratory transmitted, HIV, a retrovirus, that is mostly blood or mucosal transmitted, and canine parvovirus that is faecal:oral transmitted. We describe our current understanding of the changes in the viruses that allowed them to overcome the barriers that prevented efficient replication and spread in their new hosts. We also briefly outline how we could gain a better understanding of the mechanisms and variability in order to better anticipate these events in the future. This article is part of the theme issue 'Dynamic and integrative approaches to understanding pathogen spillover'.

Influenza viruses are important pathogens that affect multiple animal species, including humans. There are four types of influenza viruses: A, B, C, and D (IAV, IBV, ICV, and IDV, respectively). IAV and IBV are currently circulating in humans and are responsible of seasonal epidemics (IAV and IBV) and occasional pandemics (IAV). ICV is known to cause mild infections in humans and pigs, while the recently identified IDV primarily affect cattle and pigs. Influenza non-structural protein 1 (NS1) is a multifunctional protein encoded by the NS segment in all influenza types. The main function of NS1 is to counteract the host antiviral defense, including the production of interferon (IFN) and IFN-stimulated genes (ISGs), and therefore is considered an important viral pathogenic factor. Despite of homologous functions, the NS1 protein from the diverse influenza types share little amino acid sequence identity, suggesting possible differences in their mechanism(s) of action, interaction(s) with host factors, and contribution to viral replication and/or pathogenesis. In addition, although the NS1 protein of IAV, IBV and, to some extent ICV, have been previously studied, it is unclear if IDV NS1 has similar properties. Using an approach that allow us to express NS1 independently of the nuclear export protein from the viral NS segment, we have generated recombinant IAV expressing IAV, IBV, ICV, and IDV NS1 proteins. Although recombinant viruses expressing heterotypic (IBV, ICV, and IDV) NS1 proteins were able to replicate similarly in canine MDCK cells, their viral fitness was impaired in human A549 cells and they were highly attenuated in vivo. Our data suggest that despite the similarities to effectively counteract innate immune responses in vitro, the NS1 proteins of IBV, ICV, or IDV do not fully complement the functions of IAV NS1, resulting in deficient viral replication and pathogenesis in vivo.

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.

H3N8 equine influenza virus (EIV) is an important and significant respiratory pathogen of horses. EIV is enzootic in Europe and North America, mainly due to the suboptimal efficacy of current vaccines. We describe, for the first time, the generation of a temperature sensitive (ts) H3N8 EIV live-attenuated influenza vaccine (LAIV) using reverse-genetics approaches. Our EIV LAIV was attenuated (att) in vivo and able to induce, upon a single intranasal administration, protection against H3N8 EIV wild-type (WT) challenge in both a mouse model and the natural host, the horse. Notably, since our EIV LAIV was generated using reverse genetics, the vaccine can be easily updated against drifting or emerging strains of EIV using the safety backbone of our EIV LAIV as master donor virus (MDV). These results demonstrate the feasibility of implementing a novel EIV LAIV approach for the prevention and control of currently circulating H3N8 EIVs in horse populations.

Influenza's propensity for antigenic drift and shift, and to elicit predominantly strain specific antibodies (Abs) leaves humanity susceptible to waves of new strains with pandemic potential for which limited or no immunity may exist. Subsequently new clinical interventions are needed. To identify hemagglutinin (HA) epitopes that if targeted may confer universally protective humoral immunity, we examined plasmablasts from a subject that was immunized with the seasonal influenza inactivated vaccine, and isolated a human monoclonal Ab (mAb), KPF1. KPF1 has broad and potent neutralizing activity against H1 influenza viruses, and recognized 83% of all H1 isolates tested, including the pandemic 1918 H1. Prophylactically, KPF1 treatment resulted in 100% survival of mice from lethal challenge with multiple H1 influenza strains and when given as late as 72 h after challenge with A/California/04/2009 H1N1, resulted in 80% survival. KPF1 recognizes a novel epitope in the HA globular head, which includes a highly conserved amino acid, between the Ca and Cb antigenic sites. Although recent HA stalk-specific mAbs have broader reactivity, their potency is substantially limited, suggesting that cocktails of broadly reactive and highly potent HA globular head-specific mAbs, like KPF1, may have greater clinical feasibility for the treatment of influenza infections.

In 2009, a pandemic H1N1 influenza A virus (IAV) (pH1N1) emerged in the human population from swine causing a pandemic. Importantly, this virus is still circulating in humans seasonally. To analyze the evolution of pH1N1 in humans, we sequenced viral genes encoding proteins inhibiting general gene expression (nonstructural protein 1 [NS1] and PA-X) from circulating seasonal viruses and compared them to the viruses isolated at the origin of the pandemic. Recent pH1N1 viruses contain amino acid changes in the NS1 protein (E55K, L90I, I123V, E125D, K131E, and N205S), as previously described (A. M. Clark, A. Nogales, L. Martinez-Sobrido, D. J. Topham, and M. L. DeDiego, J Virol 91:e00721-17, 2017, https://doi.org/10.1128/JVI.00721-17), and amino acid changes in the PA-X protein (V100I, N204S, R221Q, and L229S). These amino acid differences between early and more recent pH1N1 isolates are responsible for increased NS1-mediated inhibition of host gene expression and decreased PA-X-mediated shutoff, including innate immune response genes. In addition, currently circulating pH1N1 viruses have acquired amino acid changes in the PA protein (V100I, P224S, N321K, I330V, and R362K). A recombinant pH1N1 virus containing PA, PA-X, and NS1 genes from currently circulating viruses is fitter in replication in cultured cells and in mice and is slightly more pathogenic than the original ancestor pH1N1 virus. These results demonstrate the need to monitor the evolution of pH1N1 in humans for mutations in the viral genome that could result in enhanced virulence. Importantly, these results further support our previous findings suggesting that inhibition of global gene expression mediated by NS1 and PA-X proteins is subject to a balance which can determine virus pathogenesis and fitness.IMPORTANCE IAVs emerge in humans from animal reservoirs, causing unpredictable pandemics. One of these pandemics was caused by an H1N1 virus in 2009, and this virus is still circulating seasonally. To analyze host-virus adaptations likely affecting influenza virus pathogenesis, protein amino acid sequences from viruses circulating at the beginning of the pandemic and those circulating currently were compared. Currently circulating viruses have incorporated amino acid changes in two viral proteins (NS1 and PA-X), affecting innate immune responses, and in the PA gene. These amino acid differences led to increased NS1-mediated and decreased PA-X-mediated inhibition of host gene expression. A recombinant pH1N1 virus containing PA, PA-X, and NS1 genes from recently circulating viruses is fitter in replication in tissue culture cells and in mice, and the virus is more pathogenic in vivo Importantly, these results suggest that a balance in the abilities of NS1 and PA-X to induce host shutoff is beneficial for IAVs.

Zika virus (ZIKV) is an emergent mosquito-borne member of the Flaviviridae family that was responsible for a recent epidemic in the Americas. ZIKV has been associated with severe clinical complications, including neurological disorder such as Guillain-Barré syndrome in adults and severe fetal abnormalities and microcephaly in newborn infants. Given the significance of these clinical manifestations, the development of tools and reagents to study the pathogenesis of ZIKV and to develop new therapeutic options are urgently needed. In this respect, the implementation of reverse genetic techniques has allowed the direct manipulation of the viral genome to generate recombinant (r)ZIKVs, which have provided investigators with powerful systems to answer important questions about the biology of ZIKV, including virus-host interactions, the mechanism of transmission and pathogenesis or the function of viral proteins. In this review, we will summarize the different reverse genetic strategies that have been implemented, to date, for the generation of rZIKVs and the applications of these platforms for the development of replicon systems or reporter-expressing viruses.

Influenza A viruses (IAV) can infect a broad range of animal hosts, including humans. In humans, IAV causes seasonal annual epidemics and occasional pandemics, representing a serious public health and economic problem, which is most effectively prevented through vaccination. The defense mechanisms that the host innate immune system provides restrict IAV replication and infection. Consequently, to successfully replicate in interferon (IFN)-competent systems, IAV has to counteract host antiviral activities, mainly the production of IFN and the activities of IFN-induced host proteins that inhibit virus replication. The IAV multifunctional proteins PA-X and NS1 are virulence factors that modulate the innate immune response and virus pathogenicity. Notably, these two viral proteins have synergistic effects in the inhibition of host protein synthesis in infected cells, although using different mechanisms of action. Moreover, the control of innate immune responses by the IAV NS1 and PA-X proteins is subject to a balance that can determine virus pathogenesis and fitness, and recent evidence shows co-evolution of these proteins in seasonal viruses, indicating that they should be monitored for enhanced virulence. Importantly, inhibition of host gene expression by the influenza NS1 and/or PA-X proteins could be explored to develop improved live-attenuated influenza vaccines (LAIV) by modulating the ability of the virus to counteract antiviral host responses. Likewise, both viral proteins represent a reasonable target for the development of new antivirals for the control of IAV infections. In this review, we summarize the role of IAV NS1 and PA-X in controlling the antiviral response during viral infection.