Dynamics of the COVID-19 epidemic in Ireland under mitigation.

BACKGROUND: In Ireland and across the European Union the COVID-19 epidemic waves, driven mainly by the emergence of new variants of the SARS-CoV-2 have continued their course, despite various interventions from governments. Public health interventions continue in their attempts to control the spread as they wait for the planned significant effect of vaccination. METHODS: To tackle this challenge and the observed non-stationary aspect of the epidemic we used a modified SEIR stochastic model with time-varying parameters, following Brownian process. This enabled us to reconstruct the temporal evolution of the transmission rate of COVID-19 with the non-specific hypothesis that it follows a basic stochastic process constrained by the available data. This model is coupled with Bayesian inference (particle Markov Chain Monte Carlo method) for parameter estimation and utilized mainly well-documented Irish hospital data. RESULTS: In Ireland, mitigation measures provided a 78-86% reduction in transmission during the first wave between March and May 2020. For the second wave in October 2020, our reduction estimation was around 20% while it was 70% for the third wave in January 2021. This third wave was partly due to the UK variant appearing in Ireland. In June 2020 we estimated that sero-prevalence was 2.0% (95% CI: 1.2-3.5%) in complete accordance with a sero-prevalence survey. By the end of April 2021, the sero-prevalence was greater than 17% due in part to the vaccination campaign. Finally we demonstrate that the available observed confirmed cases are not reliable for analysis owing to the fact that their reporting rate has as expected greatly evolved. CONCLUSION: We provide the first estimations of the dynamics of the COVID-19 epidemic in Ireland and its key parameters. We also quantify the effects of mitigation measures on the virus transmission during and after mitigation for the three waves. Our results demonstrate that Ireland has significantly reduced transmission by employing mitigation measures, physical distancing and lockdown. This has to date avoided the saturation of healthcare infrastructures, flattened the epidemic curve and likely reduced mortality. However, as we await for a full roll out of a vaccination programme and as new variants potentially more transmissible and/or more infectious could continue to emerge and mitigation measures change silent transmission, challenges remain.

A mechanistic and data-driven reconstruction of the time-varying reproduction number: Application to the COVID-19 epidemic.

The effective reproduction number Reff is a critical epidemiological parameter that characterizes the transmissibility of a pathogen. However, this parameter is difficult to estimate in the presence of silent transmission and/or significant temporal variation in case reporting. This variation can occur due to the lack of timely or appropriate testing, public health interventions and/or changes in human behavior during an epidemic. This is exactly the situation we are confronted with during this COVID-19 pandemic. In this work, we propose to estimate Reff for the SARS-CoV-2 (the etiological agent of the COVID-19), based on a model of its propagation considering a time-varying transmission rate. This rate is modeled by a Brownian diffusion process embedded in a stochastic model. The model is then fitted by Bayesian inference (particle Markov Chain Monte Carlo method) using multiple well-documented hospital datasets from several regions in France and in Ireland. This mechanistic modeling framework enables us to reconstruct the temporal evolution of the transmission rate of the COVID-19 based only on the available data. Except for the specific model structure, it is non-specifically assumed that the transmission rate follows a basic stochastic process constrained by the observations. This approach allows us to follow both the course of the COVID-19 epidemic and the temporal evolution of its Reff(t). Besides, it allows to assess and to interpret the evolution of transmission with respect to the mitigation strategies implemented to control the epidemic waves in France and in Ireland. We can thus estimate a reduction of more than 80% for the first wave in all the studied regions but a smaller reduction for the second wave when the epidemic was less active, around 45% in France but just 20% in Ireland. For the third wave in Ireland the reduction was again significant (>70%).

Stochastic Epidemic Models inference and diagnosis with Poisson Random Measure Data Augmentation.

We present a new Bayesian inference method for compartmental models that takes into account the intrinsic stochasticity of the process. We show how to formulate a SIR-type Markov jump process as the solution of a stochastic differential equation with respect to a Poisson Random Measure (PRM), and how to simulate the process trajectory deterministically from a parameter value and a PRM realization. This forms the basis of our Data Augmented MCMC, which consists of augmenting parameter space with the unobserved PRM value. The resulting simple Metropolis-Hastings sampler acts as an efficient simulation-based inference method, that can easily be transferred from model to model. Compared with a recent Data Augmentation method based on Gibbs sampling of individual infection histories, PRM-augmented MCMC scales much better with epidemic size and is far more flexible. It is also found to be competitive with Particle MCMC for moderate epidemics when using approximate simulations. PRM-augmented MCMC also yields a posteriori estimates of the PRM, that represent process stochasticity, and which can be used to validate the model. A pattern of deviation from the PRM prior distribution will indicate that the model underfits the data and help to understand the cause. We illustrate this by fitting a non-seasonal model to some simulated seasonal case count data. Applied to the Zika epidemic of 2013 in French Polynesia, our approach shows that a simple SEIR model cannot correctly reproduce both the initial sharp increase in the number of cases as well as the final proportion of seropositive. PRM augmentation thus provides a coherent story for Stochastic Epidemic Model inference, where explicitly inferring process stochasticity helps with model validation.

Parallel trends in the transmission of SARS-CoV-2 and retail/recreation and public transport mobility during non-lockdown periods.

Recent literature strongly supports the hypothesis that mobility restriction and social distancing play a crucial role in limiting the transmission of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). During the first wave of the coronavirus disease 2019 (COVID-19) pandemic, it was shown that mobility restriction reduced transmission significantly. This study found that, in the period between the first two waves of the COVID-19 pandemic, there was high positive correlation between trends in the transmission of SARS-CoV-2 and mobility. These two trends oscillated simultaneously, and increased mobility following the relaxation of lockdown rules was significantly associated with increased transmission. From a public health perspective, these results highlight the importance of tracking changes in mobility when relaxing mitigation measures in order to anticipate future changes in the spread of SARS-CoV-2.

Poultry farmer response to disease outbreaks in smallholder farming systems in southern Vietnam.

Avian influenza outbreaks have been occurring on smallholder poultry farms in Asia for two decades. Farmer responses to these outbreaks can slow down or accelerate virus transmission. We used a longitudinal survey of 53 small-scale chicken farms in southern Vietnam to investigate the impact of outbreaks with disease-induced mortality on harvest rate, vaccination, and disinfection behaviors. We found that in small broiler flocks (≤16 birds/flock) the estimated probability of harvest was 56% higher when an outbreak occurred, and 214% higher if an outbreak with sudden deaths occurred in the same month. Vaccination and disinfection were strongly and positively correlated with the number of birds. Small-scale farmers - the overwhelming majority of poultry producers in low-income countries - tend to rely on rapid sale of birds to mitigate losses from diseases. As depopulated birds are sent to markets or trading networks, this reactive behavior has the potential to enhance onward transmission.

The past few decades have seen the circulation of avian influenza viruses increase in domesticated poultry, regularly creating outbreaks associated with heavy economic loss. In addition, these viruses can sometimes 'jump' into humans, potentially allowing new diseases ­ including pandemics ­ to emerge. The Mekong river delta, in southern Vietnam, is one of the regions with the highest circulation of avian influenza. There, a large number of farmers practice poultry farming on a small scale, with limited investments in disease prevention such as vaccination or disinfection. Yet, it was unclear how the emergence of an outbreak could change the behavior of farmers. To learn more, Delabouglise et al. monitored 53 poultry farms, with fewer than 1000 chickens per farm, monthly for over a year and a half. In particular, they tracked when outbreaks occurred on each farm, and how farmers reacted. Overall, poultry farms with more than 17 chickens were more likely to vaccinate their animals and use disinfection practices than smaller farms. However, disease outbreaks did not affect vaccination or disinfection practices. When an outbreak occurred, farmers with fewer than 17 chickens tended to sell their animals earlier. For instance, they were 214% more likely to send their animals to market if an outbreak with sudden deaths occurred that month. Even if they do not make as much money selling immature individuals, this strategy may allow them to mitigate economical loss: they can sell animals that may die soon, saving on feeding costs and potentially avoiding further contamination. However, as animals were often sold alive in markets or to itinerant sellers, this practice increases the risk of spreading diseases further along the trade circuits. These data could be most useful to regional animal health authorities, which have detailed knowledge of local farming systems and personal connections in the communities where they work. This can allow them to effect change. They could work with small poultry farmers to encourage them to adopt efficient disease management strategies. Ultimately, this could help control the spread of avian influenza viruses, and potentially help to avoid future pandemics.

Poultry population dynamics and mortality risks in smallholder farms of the Mekong river delta region.

BACKGROUND: Poultry farming is widely practiced by rural households in Vietnam and the vast majority of domestic birds are kept on small household farms. However, smallholder poultry production is constrained by several issues such as infectious diseases, including avian influenza viruses whose circulation remains a threat to public health. This observational study describes the demographic structure and dynamics of small-scale poultry farms of the Mekong river delta region. METHOD: Fifty three farms were monitored over a 20-month period, with farm sizes, species, age, arrival/departure of poultry, and farm management practices recorded monthly. RESULTS: Median flock population sizes were 16 for chickens (IQR: 10-40), 32 for ducks (IQR: 18-101) and 11 for Muscovy ducks (IQR: 7-18); farm size distributions for the three species were heavily right-skewed. Muscovy ducks were kept for long periods and outdoors, while chickens and ducks were farmed indoors or in pens. Ducks had a markedly higher removal rate (broilers: 0.14/week; layer/breeders: 0.05/week) than chickens and Muscovy ducks (broilers: 0.07/week; layer/breeders: 0.01-0.02/week) and a higher degree of specialization resulting in a substantially shorter life span. The rate of mortality due to disease did not differ much among species, with birds being less likely to die from disease at older ages, but frequency of disease symptoms differed by species. Time series of disease-associated mortality were correlated with population size for Muscovy ducks (Kendall's coefficient τ = 0.49, p-value < 0.01) and with frequency of outdoor grazing for ducks (τ = 0.33, p-value = 0.05). CONCLUSION: The study highlights some challenges to disease control in small-scale multispecies poultry farms. The rate of interspecific contact and overlap between flocks of different ages is high, making small-scale farms a suitable environment for pathogens circulation. Muscovy ducks are farmed outdoors with little investment in biosecurity and few inter-farm movements. Ducks and chickens are more at-risk of introduction of pathogens through movements of birds from one farm to another. Ducks are farmed in large flocks with high turnover and, as a result, are more vulnerable to disease spread and require a higher vaccination coverage to maintain herd immunity.

Structural basis for human respiratory syncytial virus NS1-mediated modulation of host responses.

Human respiratory syncytial virus (hRSV) is a major cause of morbidity and mortality in the paediatric, elderly and immune-compromised populations1,2. A gap in our understanding of hRSV disease pathology is the interplay between virally encoded immune antagonists and host components that limit hRSV replication. hRSV encodes for non-structural (NS) proteins that are important immune antagonists3-6; however, the role of these proteins in viral pathogenesis is incompletely understood. Here, we report the crystal structure of hRSV NS1 protein, which suggests that NS1 is a structural paralogue of hRSV matrix (M) protein. Comparative analysis of the shared structural fold with M revealed regions unique to NS1. Studies on NS1 wild type or mutant alone or in recombinant RSVs demonstrate that structural regions unique to NS1 contribute to modulation of host responses, including inhibition of type I interferon responses, suppression of dendritic cell maturation and promotion of inflammatory responses. Transcriptional profiles of A549 cells infected with recombinant RSVs show significant differences in multiple host pathways, suggesting that NS1 may have a greater role in regulating host responses than previously appreciated. These results provide a framework to target NS1 for therapeutic development to limit hRSV-associated morbidity and mortality.

No Evidence of On-farm Circulation of Avian Influenza H5 Subtype in Ca Mau Province, Southern Vietnam, March 2016 - January 2017.

BACKGROUND: Subtype H5N1 avian influenza viruses, both high pathogenicity and low pathogenicity, have been enzootic in Vietnam since 2001.  The viruses are readily identified at live bird markets, but virus prevalence on smallholder poultry is typically zero or very low.  If the true direction of the viral transmission chain is farm to market, it is unknown why farm prevalence should be low when market prevalence is moderate to high. METHODS: We established a cohort of 50 smallholder poultry farms in Ca Mau province in the Mekong Delta region of Vietnam.  From March 2016 to January 2017, we collected naso-pharyngeal and cloacal samples from 156 ducks and 96 chickens.  In addition, 126 environmental samples were collected.  Samples were assayed for H5 subtype influenza by real-time RT-PCR. Results/Discussion: None of the 378 collected samples were positive for H5 influenza.  This is likely to mean that circulation of subtype H5 influenza viruses was low in Ca Mau in 2016.  Detection of avian influenza on smallholder poultry farms is necessary to determine the directionality and association between farm prevalence and market prevalence of avian influenza viruses.  Larger farm-level studies should be planned as these will be critical for determining the presence and strength of this association.

Topoisomerase II Inhibitors Induce DNA Damage-Dependent Interferon Responses Circumventing Ebola Virus Immune Evasion.

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

The Matrix Protein of Nipah Virus Targets the E3-Ubiquitin Ligase TRIM6 to Inhibit the IKKε Kinase-Mediated Type-I IFN Antiviral Response.

For efficient replication, viruses have developed mechanisms to evade innate immune responses, including the antiviral type-I interferon (IFN-I) system. Nipah virus (NiV), a highly pathogenic member of the Paramyxoviridae family (genus Henipavirus), is known to encode for four P gene-derived viral proteins (P/C/W/V) with IFN-I antagonist functions. Here we report that NiV matrix protein (NiV-M), which is important for virus assembly and budding, can also inhibit IFN-I responses. IFN-I production requires activation of multiple signaling components including the IκB kinase epsilon (IKKε). We previously showed that the E3-ubiquitin ligase TRIM6 catalyzes the synthesis of unanchored K48-linked polyubiquitin chains, which are not covalently attached to any protein, and activate IKKε for induction of IFN-I mediated antiviral responses. Using co-immunoprecipitation assays and confocal microscopy we show here that the NiV-M protein interacts with TRIM6 and promotes TRIM6 degradation. Consequently, NiV-M expression results in reduced levels of unanchored K48-linked polyubiquitin chains associated with IKKε leading to impaired IKKε oligomerization, IKKε autophosphorylation and reduced IFN-mediated responses. This IFN antagonist function of NiV-M requires a conserved lysine residue (K258) in the bipartite nuclear localization signal that is found in divergent henipaviruses. Consistent with this, the matrix proteins of Ghana, Hendra and Cedar viruses were also able to inhibit IFNß induction. Live NiV infection, but not a recombinant NiV lacking the M protein, reduced the levels of endogenous TRIM6 protein expression. To our knowledge, matrix proteins of paramyxoviruses have never been reported to be involved in innate immune antagonism. We report here a novel mechanism of viral innate immune evasion by targeting TRIM6, IKKε and unanchored polyubiquitin chains. These findings expand the universe of viral IFN antagonism strategies and provide a new potential target for development of therapeutic interventions against NiV infections.

Effects of Filovirus Interferon Antagonists on Responses of Human Monocyte-Derived Dendritic Cells to RNA Virus Infection.

UNLABELLED: Dendritic cells (DCs) are major targets of filovirus infection in vivo Previous studies have shown that the filoviruses Ebola virus (EBOV) and Marburg virus (MARV) suppress DC maturation in vitro Both viruses also encode innate immune evasion functions. The EBOV VP35 (eVP35) and the MARV VP35 (mVP35) proteins each can block RIG-I-like receptor signaling and alpha/beta interferon (IFN-α/ß) production. The EBOV VP24 (eVP24) and MARV VP40 (mVP40) proteins each inhibit the production of IFN-stimulated genes (ISGs) by blocking Jak-STAT signaling; however, this occurs by different mechanisms, with eVP24 blocking nuclear import of tyrosine-phosphorylated STAT1 and mVP40 blocking Jak1 function. MARV VP24 (mVP24) has been demonstrated to modulate host cell antioxidant responses. Previous studies demonstrated that eVP35 is sufficient to strongly impair primary human monocyte-derived DC (MDDC) responses upon stimulation induced through the RIG-I-like receptor pathways. We demonstrate that mVP35, like eVP35, suppresses not only IFN-α/ß production but also proinflammatory responses after stimulation of MDDCs with RIG-I activators. In contrast, eVP24 and mVP40, despite suppressing ISG production upon RIG-I activation, failed to block upregulation of maturation markers or T cell activation. mVP24, although able to stimulate expression of antioxidant response genes, had no measurable impact of DC function. These data are consistent with a model where filoviral VP35 proteins are the major suppressors of DC maturation during filovirus infection, whereas the filoviral VP24 proteins and mVP40 are insufficient to prevent DC maturation. IMPORTANCE: The ability to suppress the function of dendritic cells (DCs) likely contributes to the pathogenesis of disease caused by the filoviruses Ebola virus and Marburg virus. To clarify the basis for this DC suppression, we assessed the effect of filovirus proteins known to antagonize innate immune signaling pathways, including Ebola virus VP35 and VP24 and Marburg virus VP35, VP40, and VP24, on DC maturation and function. The data demonstrate that the VP35s from Ebola virus and Marburg virus are the major suppressors of DC maturation and that the effects on DCs of the remaining innate immune inhibitors are minor.

Differential Regulation of Interferon Responses by Ebola and Marburg Virus VP35 Proteins.

Suppression of innate immune responses during filoviral infection contributes to disease severity. Ebola (EBOV) and Marburg (MARV) viruses each encode a VP35 protein that suppresses RIG-I-like receptor signaling and interferon-α/ß (IFN-α/ß) production by several mechanisms, including direct binding to double stranded RNA (dsRNA). Here, we demonstrate that in cell culture, MARV infection results in a greater upregulation of IFN responses as compared to EBOV infection. This correlates with differences in the efficiencies by which EBOV and MARV VP35s antagonize RIG-I signaling. Furthermore, structural and biochemical studies suggest that differential recognition of RNA elements by the respective VP35 C-terminal IFN inhibitory domain (IID) rather than affinity for RNA by the respective VP35s is critical for this observation. Our studies reveal functional differences in EBOV versus MARV VP35 RNA binding that result in unexpected differences in the host response to deadly viral pathogens.

Prothymosin α variants isolated from CD8+ T cells and cervicovaginal fluid suppress HIV-1 replication through type I interferon induction.

Soluble factors from CD8(+) T cells and cervicovaginal mucosa of women are recognized as important in controlling human immunodeficiency virus type 1 (HIV-1) infection and transmission. Previously, we have shown the strong anti-HIV-1 activity of prothymosin α (ProTα) derived from CD8(+) T cells. ProTα is a small acidic protein with wide cell distribution, to which several functions have been ascribed, depending on its intracellular or extracellular localization. To date, activities of ProTα have been attributed to a single protein known as isoform 2. Here we report the isolation and identification of 2 new ProTα variants from CD8(+) T cells and cervicovaginal lavage with potent anti-HIV-1 activity. The first is a splice variant of the ProTα gene, known as isoform CRA_b, and the second is the product of a ProTα gene, thus far classified as a pseudogene 7. Native or recombinant ProTα variants potently restrict HIV-1 replication in macrophages through the induction of type I interferon. The baseline expression of interferon-responsive genes in primary human cervical tissues positively correlate with high levels of intracellular ProTα, and the knockdown of ProTα variants by small interfering RNA leads to downregulation of interferon target genes. Overall, these findings suggest that ProTα variants are innate immune mediators involved in immune surveillance.

Ebola virus VP24 targets a unique NLS binding site on karyopherin alpha 5 to selectively compete with nuclear import of phosphorylated STAT1.

During antiviral defense, interferon (IFN) signaling triggers nuclear transport of tyrosine-phosphorylated STAT1 (PY-STAT1), which occurs via a subset of karyopherin alpha (KPNA) nuclear transporters. Many viruses, including Ebola virus, actively antagonize STAT1 signaling to counteract the antiviral effects of IFN. Ebola virus VP24 protein (eVP24) binds KPNA to inhibit PY-STAT1 nuclear transport and render cells refractory to IFNs. We describe the structure of human KPNA5 C terminus in complex with eVP24. In the complex, eVP24 recognizes a unique nonclassical nuclear localization signal (NLS) binding site on KPNA5 that is necessary for efficient PY-STAT1 nuclear transport. eVP24 binds KPNA5 with very high affinity to effectively compete with and inhibit PY-STAT1 nuclear transport. In contrast, eVP24 binding does not affect the transport of classical NLS cargo. Thus, eVP24 counters cell-intrinsic innate immunity by selectively targeting PY-STAT1 nuclear import while leaving the transport of other cargo that may be required for viral replication unaffected.

Molecular basis for ebolavirus VP35 suppression of human dendritic cell maturation.

UNLABELLED: Zaire ebolavirus (EBOV) VP35 is a double-stranded RNA (dsRNA)-binding protein that inhibits RIG-I signaling and alpha/beta interferon (IFN-α/ß) responses by both dsRNA-binding-dependent and -independent mechanisms. VP35 also suppresses dendritic cell (DC) maturation. Here, we define the pathways and mechanisms through which VP35 impairs DC maturation. Wild-type VP35 (VP35-WT) and two well-characterized VP35 mutants (F239A and R322A) that independently ablate dsRNA binding and RIG-I inhibition were delivered to primary human monocyte-derived DCs (MDDCs) using a lentivirus-based expression system. VP35-WT suppressed not only IFN-α/ß but also proinflammatory responses following stimulation of MDDCs with activators of RIG-I-like receptor (RLR) signaling, including RIG-I activators such as Sendai virus (SeV) or 5'-triphosphate RNA, or MDA5 activators such as encephalomyocarditis virus (EMCV) or poly(I · C). The F239A and R322A mutants exhibited greatly reduced suppression of IFN-α/ß and proinflammatory cytokine production following treatment of DCs with RLR agonists. VP35-WT also blocked the upregulation of DC maturation markers and the stimulation of allogeneic T cell responses upon SeV infection, whereas the mutants did not. In contrast to the RLR activators, VP35-WT and the VP35 mutants impaired IFN-ß production induced by Toll-like receptor 3 (TLR3) or TLR4 agonists but failed to inhibit proinflammatory cytokine production induced by TLR2, TLR3, or TLR4 agonists. Furthermore, VP35 did not prevent lipopolysaccharide (LPS)-induced upregulation of surface markers of MDDC maturation and did not prevent LPS-triggered allogeneic T cell stimulation. Therefore, VP35 is a general antagonist of DC responses to RLR activation. However, TLR agonists can circumvent many of the inhibitory effects of VP35. Therefore, it may be possible to counteract EBOV immune evasion by using treatments that bypass the VP35-imposed block to DC maturation. IMPORTANCE: The VP35 protein, which is an inhibitor of RIG-I signaling and alpha/beta interferon (IFN-α/ß) responses, has been implicated as an EBOV-encoded factor that contributes to suppression of dendritic cell (DC) function. We used wild-type VP35 and previously characterized VP35 mutants to clarify VP35-DC interactions. Our data demonstrate that VP35 is a general inhibitor of RIG-I-like receptor (RLR) signaling that blocks not only RIG-I- but also MDA5-mediated induction of IFN-α/ß responses. Furthermore, in DCs, VP35 also impairs the RLR-mediated induction of proinflammatory cytokine production, upregulation of costimulatory markers, and activation of T cells. These inhibitory activities require VP35 dsRNA-binding activity, an activity previously correlated to VP35 RIG-I inhibitory function. In contrast, while VP35 can inhibit IFN-α/ß production induced by TLR3 or TLR4 agonists, this occurs in a dsRNA-independent fashion, and VP35 does not inhibit TLR-mediated expression of proinflammatory cytokines. These data suggest strategies to overcome VP35 inhibition of DC function.

Mutual antagonism between the Ebola virus VP35 protein and the RIG-I activator PACT determines infection outcome.

The cytoplasmic pattern recognition receptor RIG-I is activated by viral RNA and induces type I IFN responses to control viral replication. The cellular dsRNA binding protein PACT can also activate RIG-I. To counteract innate antiviral responses, some viruses, including Ebola virus (EBOV), encode proteins that antagonize RIG-I signaling. Here, we show that EBOV VP35 inhibits PACT-induced RIG-I ATPase activity in a dose-dependent manner. The interaction of PACT with RIG-I is disrupted by wild-type VP35, but not by VP35 mutants that are unable to bind PACT. In addition, PACT-VP35 interaction impairs the association between VP35 and the viral polymerase, thereby diminishing viral RNA synthesis and modulating EBOV replication. PACT-deficient cells are defective in IFN induction and are insensitive to VP35 function. These data support a model in which the VP35-PACT interaction is mutually antagonistic and plays a fundamental role in determining the outcome of EBOV infection.

Ebola virus exploits a monocyte differentiation program to promote its entry.

Antigen-presenting cells (APCs) are critical targets of Ebola virus (EBOV) infection in vivo. However, the susceptibility of monocytes to infection is controversial. Studies indicate productive monocyte infection, and yet monocytes are also reported to be resistant to EBOV GP-mediated entry. In contrast, monocyte-derived macrophages and dendritic cells are permissive for both EBOV entry and replication. Here, freshly isolated monocytes are demonstrated to indeed be refractory to EBOV entry. However, EBOV binds monocytes, and delayed entry occurs during monocyte differentiation. Cultured monocytes spontaneously downregulate the expression of viral entry restriction factors such as interferon-inducible transmembrane proteins, while upregulating the expression of critical EBOV entry factors cathepsin B and NPC1. Moreover, these processes are accelerated by EBOV infection. Finally, ectopic expression of NPC1 is sufficient to rescue entry into an undifferentiated, normally nonpermissive monocytic cell line. These results define the molecular basis for infection of APCs and suggest means to limit APC infection.

In vivo expansion of regulatory T cells with IL-2/IL-2 mAb complexes prevents anti-factor VIII immune responses in hemophilia A mice treated with factor VIII plasmid-mediated gene therapy.

Generation of transgene-specific immune responses can constitute a major complication following gene therapy treatment. An in vivo approach to inducing selective expansion of Regulatory T (Treg) cells by injecting interleukin-2 (IL-2) mixed with a specific IL-2 monoclonal antibody (JES6-1) was adopted to modulate anti-factor VIII (anti-FVIII) immune responses. Three consecutive IL-2 complexes treatments combined with FVIII plasmid injection prevented anti-FVIII formation and achieved persistent, therapeutic-level of FVIII expression in hemophilia A (HemA) mice. The IL-2 complexes treatment expanded CD4(+)CD25(+)Foxp3(+) Treg cells five- to sevenfold on peak day, and they gradually returned to normal levels within 7-14 days without changing other lymphocyte populations. The transiently expanded Treg cells are highly activated and display suppressive function in vitro. Adoptive transfer of the expanded Treg cells protected recipient mice from generation of high-titer antibodies following FVIII plasmid challenge. Repeated plasmid transfer is applicable in tolerized mice without eliciting immune responses. Mice treated with IL-2 complexes mounted immune responses against both T-dependent and T-independent neoantigens, indicating that IL-2 complexes did not hamper the immune system for long. These results demonstrate the important role of Treg cells in suppressing anti-FVIII immune responses and the potential of developing Treg cell expansion therapies that induce long-term tolerance to FVIII.

CD4+FOXP3+ regulatory T cells confer long-term regulation of factor VIII-specific immune responses in plasmid-mediated gene therapy-treated hemophilia mice.

Gene transfer of a factor VIII (FVIII) plasmid into hemophilia A (HemA) mice achieved supraphysiologic FVIII expression, but triggered production of high-titer FVIII-specific antibodies and loss of functional FVIII activity. To test whether FVIII-specific regulatory T cells (Tregs) can modulate immune responses against FVIII, we developed a HemA mouse model in which all T cells overexpressed Foxp3 (HemA/Foxp3-Tg). FVIII plasmid therapy did not induce antibody production in HemA/Foxp3-Tg mice. CD4(+)Foxp3(+) T cells isolated from plasmid-treated HemA/Foxp3-Tg mice significantly suppressed proliferation of FVIII-stimulated CD4(+) effector T cells. The percentage of CD4(+) T cells expressing CD25, glucocorticoid-induced tumor necrosis factor receptor, and cytotoxic T lymphocyte antigen 4 increased significantly in spleen and peripheral blood for 9 weeks. Mice receiving adoptively transferred Tregs from FVIII-exposed HemA/Foxp3-Tg mice produced significantly reduced antibody titers compared with controls after initial challenge with FVIII plasmid and second challenge 16 weeks after first plasmid treatment. Adoptively transferred Tregs engrafted and distributed at 2% to 4% in the Treg compartment of blood, lymph nodes, and spleens of the recipient mice and induced activation of endogenous Tregs; the engraftment decreased to negligible levels over 8 to 12 weeks. Antigen-specific Tregs can provide long-lasting protection against immune responses in vivo and limit recall responses induced by a second challenge via infectious tolerance.

ABIN-1 is a ubiquitin sensor that restricts cell death and sustains embryonic development.

Proteins that directly regulate tumour necrosis factor receptor (TNFR) signalling have critical roles in regulating cellular activation and survival. ABIN-1 (A20 binding and inhibitor of NF-kappaB) is a novel protein that is thought to inhibit NF-kappaB signalling. Here we show that mice deficient for ABIN-1 die during embryogenesis with fetal liver apoptosis, anaemia and hypoplasia. ABIN-1 deficient cells are hypersensitive to tumour necrosis factor (TNF)-induced programmed cell death, and TNF deficiency rescues ABIN-1 deficient embryos. ABIN-1 inhibits caspase 8 recruitment to FADD (Fas-associated death domain-containing protein) in TNF-induced signalling complexes, preventing caspase 8 cleavage and programmed cell death. Moreover, ABIN-1 directly binds polyubiquitin chains and this ubiquitin sensing activity is required for ABIN-1's anti-apoptotic activity. These studies provide insights into how ubiquitination and ubiquitin sensing proteins regulate cellular and organismal survival.