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Poxviruses and the immune system: Implications for monkeypox virus
Abstract
Poxviruses (PXVs) are mostly known for the variola virus, being the cause of smallpox; however, re-emerging PXVs have also shown a great capacity to develop outbreaks of pox-like infections in humans. The situation is alarming; PXV outbreaks have been involving both endemic and non-endemic areas in recent decades. Stopped smallpox vaccination is a reason offered mainly for this changing epidemiology that implies the protective role of immunity in the pathology of PXV infections. The immune system recognizes PXVs and elicits responses, but PXVs can antagonize these responses. Here, we briefly review the immunology of PXV infections, with emphasis on the role of pattern-recognition receptors, macrophages, and natural killer cells in the early response to PXV infections and PXVs’ strategies influencing these responses, as well as taking a glance at other immune cells, which discussion over them mainly occurs in association with PXV immunization rather than PXV infection. Throughout the review, numerous evasion mechanisms are highlighted, which might have implications for designing specific immunotherapies for PXV in the future.
... The feature of innate immunity lies in the presence of pattern recognition receptors (PRRs) which are used to initiate an immune response cascade [56,76], and this represents the first line of defense following active viral infection [59]. Many cells play key roles in this immune response such as natural killer cells which modulate the functions of other immunity cells like T cells through cytokines secretions [36,58,59], and dendritic cells (antigen-presenting cells) which migrate to viral replication sites to activate other immune cells [77]. Moreover, this complex system performs a variety of functions, including the removal of cellular debris, the initiation of an inflammatory response, the activation of adaptive immunity, and the recognition of virus-infected cells [63]. ...
... Interferon-gamma (IFN-γ) and Tumor Necrosis Factor α (TNF-α), or directly through cell-to-cell contact [77]. Subsequently, the second stage of immune defense takes place under the surveillance of B cells which differentiate into plasma cells that in turn, migrate to immune organs like the lymph nodes, mucus membranes, and bone marrow to produce specific antibodies against MPXV and facilitate their entry to the bloodstream to control the viral spread [77,79]. ...
... Interferon-gamma (IFN-γ) and Tumor Necrosis Factor α (TNF-α), or directly through cell-to-cell contact [77]. Subsequently, the second stage of immune defense takes place under the surveillance of B cells which differentiate into plasma cells that in turn, migrate to immune organs like the lymph nodes, mucus membranes, and bone marrow to produce specific antibodies against MPXV and facilitate their entry to the bloodstream to control the viral spread [77,79]. ...
A new emerging virus named Monkeypox virus (MPXV) has recently gained significant attention, captured the headlines and spread panic among people due to the threat it poses to their health and well-being. This review paper aims to provide an overview of the virology of this novel virus, shedding light on its morphology, genetics, and process of replication. Furthermore, it delves into the origin of the virus and describes the diverse routes through which it spreads, as well as our body's immune response against it. Understanding the fundamental characteristics of the MPXV is crucial in order to comprehend its potential impact on public health. Also, exploring its genetics, and studying its replication would aid in the development of effective diagnostic methods and potential treatments, and to identify specific genes and proteins that contribute to its virulence and pathogenicity. While the likelihood of MPXV becoming a pandemic may be low, comprehensive knowledge about the virus and its modes of transmission is crucial to prevent the possibility of large-scale outbreaks. By staying informed and implementing appropriate preventive measures, we can effectively mitigate the impact of this emerging virus and safeguard public health.
... IEVs undergo fusion with the plasma membrane, resulting in the formation of a cell-associated enveloped virus [48], which can either be removed from the cell (extruded cell) or released as an extracellular enveloped virus (EEV). It is also possible for the MVs to bud directly and release the EEV, thereby bypassing the formation of the IEV [49]. ...
... The host's immune response to mpox infection involves the activation of innate immune cells such as natural killer cells and inflammatory cells and the production of interferons (IFNs) and the complement system, which work together to clear the virus and facilitate cell and humoral responses, including antibody production and viral clearance mechanisms. However, despite these immune mechanisms, mpox has developed immune evasion strategies to bypass the host's defense and establish a pathogenic infection [49]. ...
... However, poxviruses have been shown to interfere with this process by using semaphorin family proteins that bind to the receptor Plexin C1. This interaction inhibits phagocytosis in neutrophils and dendritic cells [56], which is involved in the immunopathogenesis of monkeypox [49]. ...
Monkeypox virus (MPXV) is an emerging zoonotic virus that belongs to the Orthopoxvirus genus and presents clinical symptoms similar to those of smallpox, such as fever and vesicular–pustular skin lesions. However, the differential diagnosis between smallpox and monkeypox is that smallpox does not cause lymphadenopathy but monkeypox generates swelling in the lymph nodes. Since the eradication of smallpox, MPXV has been identified as the most common Orthopoxvirus to cause human disease. Despite MPXV being endemic to certain regions of Africa, the current MPXV outbreak, which began in early 2022, has spread to numerous countries worldwide, raising global concern. As of the end of May 2023, over 87,545 cases and 141 deaths have been reported, with most cases identified in non-endemic countries, primarily due to human-to-human transmission. To better understand this emerging threat, this review presents an overview of key aspects of MPXV infection, including its animal reservoirs, modes of transmission, animal models, epidemiology, clinical and immunological features, diagnosis, treatments, vaccines, and prevention strategies. The material presented here provides a comprehensive understanding of MPXV as a disease, while emphasizing the significance and unique characteristics of the 2022 outbreak. This offers valuable information that can inform future research and aid in the development of effective interventions.
... Studies confirm that NK cells are infected with MPXV, are often associated with a Th1 response, NK-cell activation, IFN-γ production, and T-cell activation [18]. TGF-β1 suppresses the functions of Th1 and Th2, NK cells, and CD4+ effector cells, and promotes generation of Treg cells. ...
... Some authors suggest that the initial development of Mpox lesions at the genital, perianal, and perioral or tonsillar regions, with a history of recent sexual contact, may be an indication of the initial site of inoculation. 18 Symptoms, in this case, resolved within three weeks of the onset of symptoms on conservative management without the patient requiring hospitalization, this is a reflection of the fact that Mpox disease is in most cases a self-limiting [18]. This case was also quickly contained due to the efficiency of the experienced Rivers PHEOC. ...
... Some authors suggest that the initial development of Mpox lesions at the genital, perianal, and perioral or tonsillar regions, with a history of recent sexual contact, may be an indication of the initial site of inoculation. 18 Symptoms, in this case, resolved within three weeks of the onset of symptoms on conservative management without the patient requiring hospitalization, this is a reflection of the fact that Mpox disease is in most cases a self-limiting [18]. This case was also quickly contained due to the efficiency of the experienced Rivers PHEOC. ...
Mpox presents typically with macular papular body rashes with similar-looking sores in genital or oral areas. It may be missed, or wrongly managed as sexually transmitted infections (STI), especially in settings where symptom-based treatment practices are sufficient for care. We describe a 40-year-old cisgender male, heterosexual Nigerian who developed some rash in the genital region associated with penile swelling. It was suspected to be syphilis and managed with presumptive antibiotics. Laboratory investigation (PCR) confirmed that it was a Mpox disease, and Case Study Owhonda et al.; Arch. Curr. Res. Int., vol. 23, no. 2, pp. 36-42, 2023; Article no.ACRI.97058 37 VDRLT was negative. Notably, in this case, the early symptoms were a paradigm case of Mpox disease presenting as an STI, which syndromically conformed more to an STI than the case definition for a suspected case of Mpox in our setting. Consequently, the expected early containment activities were delayed. This increased the potential of further disease spread. An Update of the standard case definition as well as a reclassification of the disease as a possible STI is recommended for enhanced surveillance, increased case detection, and reduction in the burden of unrecognized cases.
... Many researches have been conducted on these viruses and their pathogenic mechanisms and have shown that Mpox has many structural and functional similarities with other orthopoxviruses. For this reason, the interaction of Mpox with the human immune system is inferred from studies conducted on VACV [30][31][32]. Mammalian cells have developed PRRs to detect microbial pathogens, including viral nucleic acids. ...
... Research has shown that direct Toll-like receptor 2 (TLR2) signaling is important and necessary to stimulate NK cells during Mpox infection [70]. In the activation and function of NK cells, Mpox viruses can directly interact with TLR2 on NK cells, leading to their activation and subsequent immune response [31]. In addition, the NKG2D receptor is involved in NK cell resistance to Mpox disease [71]. ...
Background: The reemergence of monkeypox virus (Mpox, formerly monkeypox) in 2022 in non-endemic countries has raised significant concerns for global health due to its high transmissibility and mortality rate. A major challenge in combating Mpox is its ability to evade the host’s innate immune system, the first line of defense against viral infections. Summary: Mpox encodes various proteins that interfere with key antiviral pathways and mechanisms, such as the nuclear factor kappa B signaling, cytokine production, complement and inflammasome activation, and chemokine binding. These proteins modulate the expression and function of innate immune mediators, such as interferons, interleukins, and Toll-like receptors, and impair the recruitment and activation of innate immune cells, such as natural killer cells. By suppressing or altering these innate immune responses, Mpox enhances its replication and infection in the host tissues and organs, leading to systemic inflammation, tissue damage, and organ failure. Key Messages: This study reveals new insights into the molecular and cellular interactions between Mpox and the host’s innate immune system. It identifies potential targets and strategies for antiviral interventions, highlighting the importance of understanding these interactions to develop effective treatments and improve global health responses to Mpox outbreaks.
... The importance of B-cell-mediated immune responses in the form of the production of specific antibodies is of prime importance due to the successful vaccination of smallpox in history based upon B-cell-mediated immunity [43]. Some studies have suggested that treatment with vaccine immune globulin (VIG) could save patients from mpox infection [44,45]. ...
... Some studies have suggested that treatment with vaccine immune globulin (VIG) could save patients from mpox infection [44,45]. This hypothesis was checked in monkey models and showed that VACV-specific B-cell responses could protect against a lethal mpox infection [43]. This sort of inference is beneficial for the scientific community designing vaccines along similar lines to the VACV vaccine or using VACV against mpox infection to check the results [44]. ...
Background: The years 2022-2023 witnessed a monkeypox virus (mpox) outbreak in some countries worldwide, where it exists in an endemic form. However, the number of infectious cases is continuously on the rise, and there has been an unexpected, drastic increase in cases that result from sustained transmission in non-endemic regions of the world. Under this scenario, it is pertinent for the world to be aware of healthcare threats to mpox infection. This review aimed to compile advanced data regarding the different aspects of mpox disease. Methods: A comprehensive strategy for the compilation of recent data was adopted to add data regarding mpox, biology, viral pathology, immune response, and brief details on the antiviral strategies under trial; the search was limited to 2016-2023. The aim is to make the scientific community aware of diverse aspects of mpox. Results: Consequently, detailed insights have been drawn with regard to the nature, epidemiology, etiology, and biological nature of mpox. Additionally, its host interaction and viral infectious cycle and immune interventions have been briefly elaborated. This comprehensively drawn literature review delivers brief insights into the biological nature, immune responses, and clinical developments in the form of therapeutics against mpox. This study will help scientists understand the biological nature and responses in hosts, which will further help clinicians with therapeutic handling, diagnosis, and treatment options. Conclusions: This study will provide updated information on mpox's pathology, immune responses, and antiviral strategies. Moreover, it will also help the public to become educated on the healthcare-associated threat and take timely mitigation measures against expected mpox outbreaks in the future.
... The importance of B-cell-mediated immune responses in the form of the production of specific antibodies is of prime importance due to the successful vaccination of smallpox in history based upon B-cell-mediated immunity [43]. Some studies have suggested that treatment with vaccine immune globulin (VIG) could save patients from mpox infection [44,45]. ...
... Some studies have suggested that treatment with vaccine immune globulin (VIG) could save patients from mpox infection [44,45]. This hypothesis was checked in monkey models and showed that VACV-specific B-cell responses could protect against a lethal mpox infection [43]. This sort of inference is beneficial for the scientific community designing vaccines along similar lines to the VACV vaccine or using VACV against mpox infection to check the results [44]. ...
Background:
The years 2022-2023 witnessed a monkeypox virus (mpox) outbreak in some countries worldwide, where it exists in an endemic form. However, the number of infectious cases is continuously on the rise, and there has been an unexpected, drastic increase in cases that result from sustained transmission in non-endemic regions of the world. Under this scenario, it is pertinent for the world to be aware of healthcare threats to mpox infection. This review aimed to compile advanced data regarding the different aspects of mpox disease.
Methods:
A comprehensive strategy for the compilation of recent data was adopted to add data regarding mpox, biology, viral pathology, immune response, and brief details on the antiviral strategies under trial; the search was limited to 2016-2023. The aim is to make the scientific community aware of diverse aspects of mpox.
Results:
Consequently, detailed insights have been drawn with regard to the nature, epidemiology, etiology, and biological nature of mpox. Additionally, its host interaction and viral infectious cycle and immune interventions have been briefly elaborated. This comprehensively drawn literature review delivers brief insights into the biological nature, immune responses, and clinical developments in the form of therapeutics against mpox. This study will help scientists understand the biological nature and responses in hosts, which will further help clinicians with therapeutic handling, diagnosis, and treatment options.
Conclusions:
This study will provide updated information on mpox's pathology, immune responses, and antiviral strategies. Moreover, it will also help the public to become educated on the healthcare-associated threat and take timely mitigation measures against expected mpox outbreaks in the future.
... The Mpox virus has developed complex evasion tactics to avoid immune recognition and create a prolonged infection in the host [42,43]. These evasion strategies target different components of the host immune response, allowing the virus to circumvent antiviral defenses and boost its survival and spread. ...
Background and Aim: Monkeypox (Mpox) is a viral disease mainly found in central and western Africa, with symptoms similar to variola virus (smallpox) but distinguished by the early lymph node swelling specific to Mpox. This review summarizes the neuropsychiatric manifestations of Mpox infection and vaccination, along with management approaches. Method: We searched different databases such as PubMed, Scopus, WoS, and Google Scholar about the neuropsychiatric manifestations of Mpox disease and the associated strategies of management. Results and conclusions: Mpox can cause a wide range of neurological symptoms. These range from mild symptoms like headaches, muscle aches, fatigue, and pain to severe symptoms, including seizures, blindness, photophobia, delirium, coma, encephalitis, and transverse myelitis. It is essential to distinguish Mpox from smallpox and other orthopox viruses. Psychiatric issues, such as stigma, disfigurement, isolation, and physical pain, are common in Mpox patients. To address these, healthcare providers should provide accurate information, counseling, and virtual support. Neurological side effects were associated with the previous smallpox vaccine, which offered cross-protection against Mpox. This vaccine has since been replaced by JYNNEOS, which does not pose any neurological risks. Mpox-related neurological symptoms are generally managed with supportive care, including NSAIDs, antibiotics, antiepileptics, and sedatives for seizures. Antivirals like acyclovir are also used. Severe cases may require hospitalization or intubation. So, we recommend early diagnosis, isolation, and prompt treatment, as Mpox spreading to the central nervous system can lead to serious and potentially fatal complications.
... What are the innate and adaptive immune responses to MPVX?5.2.1 | Innate Immune Responses to MPXV[95][96][97][98][99][100][101] The first line of defense, innate immunity, develops at the beginning of an active viral infection in the body. Interestingly, the very early innate immune response provides a viral entry route. ...
As the world still vividly recalls the previous monkeypox (mpox) outbreak that impacted over 120 countries worldwide with more than 99,000 cases in 2022, we are now facing a second wave of infections from the monkeypox virus (MPXV), characterized by an exponential increase in cases. The current 2024 outbreak has already recorded more than 20,000 cases in Africa, marking a dramatic escalation compared to previous outbreaks. The predominance of the newly identified clade Ib variant, first detected in the Democratic Republic of the Congo (DRC) and now identified across multiple African nations and beyond, underscores its enhanced transmissibility and potential for international spread, evidenced by cases in Sweden and Thailand. The World Health Organization (WHO) declared on August 14, 2024, the current mpox outbreak a Public Health Emergency of International Concern (PHEIC), calling for heightened global public health measures. The ongoing pattern of unusual, frequent, and extensive outbreaks of mpox with potential global implications poses significant questions. This review addresses, in the format of 50 questions and answers, the 2024 mpox outbreak, detailing its characteristics, epidemiological data, and impact compared to previous outbreaks. It comprehensively explores critical questions related to MPXV virological characteristics, immunological response, clinical manifestations, epidemiology, diagnostics, and available treatments. The review also documents the significant and evolving challenges posed by the current mpox outbreak, highlighting its scale, spread, and public health response.
... The central part of the MPXV genome that codes for crucial enzymes and structural proteins is 96.3% identical to variola virus (VARVs) [3]. MPXV's many relatives cause rodent infection (cowpox, ectromelia, variola) [4]. The taxonomy of MPXV is shown in Table 1. ...
Purpose of review
Monkeypox (Mpox) is a zoonotic disease caused by monkeypox virus (MPXV) was formerly restricted within western and central Africa, but a surge in the number of cases in 2022, 2023 and 2024 is seen in several nations across the globe. This review gives a comprehensive update on the epidemiology of Mpox, MPXV reservoirs, transmission characteristics, mutation, mechanisms of infection, clinical indications, laboratory diagnosis, ways of management, and potential vaccine candidates.
Recent findings
WHO announced the Mpox outburst as a public health emergency of international concern (PHEIC) on 14th August 2024 as a new variant of Mpox is reported and cases are now being documented for the first time from nations like Kenya and Rwanda. Recent cases specify that inter-human transmission might result in frequent eruptions, particularly in men who are sexually active with men. The creation of antiviral medicines and vaccines for treating Mpox is immediately required, regardless of the specific beneficial effects of repurposed drugs.
Summary
Mpox is a viral disease responsible for symptoms such as rash, critical lymphadenopathy, and fever. In this article, we discussed preventive approaches like immunization using smallpox vaccines, providing insights on proper understanding of MPXV and suggestions for the management, prevention, and control of MPXV.
... Direct neural invasion emerges as a significant mechanism facilitating poxvirus entry into the CNS, resulting in conditions such as encephalitis or myelitis [31]. VACV employed in smallpox vaccination, has been documented to infect peripheral nerves and retrogradely transport to the CNS, instigating neurological complications [32]. ...
Poxviridae is a diverse family of double-stranded DNA viruses, historically significant for diseases like smallpox caused by variola virus (VARV). These viruses exhibit unique cytoplasmic replication strategies, large genomes encoding numerous proteins, and the ability to cause severe cutaneous and systemic diseases. Recent attention has focused on their neurotropic potential, including mechanisms of CNS invasion, immune-mediated damage, and clinical manifestations such as encephalitis and myelitis. This review synthesises current knowledge on poxvirus neurotropism, highlighting pathophysiological mechanisms and clinical implications.
... Innate immune cells typically represent the first defense against viruses; however, these cells are also targets for MPXV, facilitating virus dissemination in the host. Consequently, the detection of MPXV antigens in innate cells such as monocytes and neutrophils has been associated with MPXV lethality [30][31][32][33]. The cells intended to fight the infection act as Trojan horses and induce the immune-specific response, including the release of several chemokines and cytokines that amplify the response and recruit more neutrophils and monocytes at the site of infection. ...
Background:
Monkeypox (Mpox) is a zoonotic disease caused by monkeypox virus (MPXV), an Orthopoxvirus (OPXV). Since we are observing the first MPXV outbreak outside the African continent, the general population probably does not have a pre-existing memory response for MPXV but may have immunity against the previous smallpox vaccine based on a live replicating Vaccinia strain (VACV). Using a whole blood platform, we aim to study the MPXV- T-cell-specific response in Mpox-cured subjects.
Methods:
We enrolled 16 subjects diagnosed with Mpox in the previous 3-7 months and 15 healthy donors (HD) with no recent vaccination history. Whole blood was stimulated overnight with MPXV and VACV peptides to elicit CD4 and CD8 T-cell-specific responses, which were evaluated by ELISA and multiplex assay.
Results:
Mpox-cured subjects showed a significant IFN-γ T-cell response to MPXV and VACV. Besides IFN-γ, IL-6, IP-10, IL-8, IL-2, G-CSF, MCP-1, MIP1-α, MIP-1β, IL-1Rα, and IL-5 were significantly induced after specific stimulation compared to the unstimulated control. The specific response was mainly induced by the CD4 peptides MPX-CD4-E and VACV-CD4.
Conclusions:
We showed that MPXV-specific responses have a mixed Th1- and Th2-response in a whole blood platform assay, which may be useful for monitoring the specific immunity induced by vaccination or infection.
... Accessed on March 16, 2023). Monkeypox, cowpox, camelpox, ectromelia, myxoma, rabbit fibroma, vaccinia, contagious ecthyma, sheeppox, goatpox, swinepox, bovine papular stomatitis, pseudocowpox, lumpy skin disease, and fowlpox are various diseases caused by pox viruses (De Clercq 2002;Haller et al. 2014;Saghazadeh and Rezaei 2022). Since the 1950s, vaccinia virus has been used as a model to developing anti-poxviral drugs. ...
Viral diseases pose significant challenges in human and veterinary medicine, as effective treatment options are limited. The development of antiviral drugs becomes increasingly challenging in the face of emerging and reemerging viral diseases. The genetic changes and rapid evolution of certain viruses further complicate the development of universal pathogen-specific drug therapies. Hence, there is a pressing need for research and development in the field of antiviral drugs. The novel approaches, such as computer-aided drug designing (CADD), have expedited the drug development process, notably reducing the preclinical phase. Phyto-medicines offer promising alternatives as antivirals, while organic polymers like extracellular polymeric substances (EPS) have demonstrated effectiveness in combating viruses. Additionally, exosomes have shown potential as antivirals and in antiviral delivery, particularly when loaded with drugs and specifically targeted against viral diseases. The repurposing of drugs for antiviral therapeutics has garnered significant attention in both research institutions and pharmaceutical industries due to its numerous advantages. Combination antiviral therapies not only reduce toxicity but also address issues of drug resistance. Lastly, extensive clinical trials will be crucial for the successful repurposing of drugs as antiviral therapeutics.
... The immune response to MPXV is a complex interplay involving both innate and adaptive immunity. 11,18 Innate Immunity: This is the body's first line of defense against MPXV. Upon infection, the host's innate immune system recognizes the virus through pattern recognition receptors (PRRs) that identify pathogen-associated molecular patterns (PAMPs) on the virus. ...
Monkeypox virus (MPXV), a member of the Orthopoxvirus genus, shares its genus with Variola virus (VARV), the causative agent of smallpox, and Vaccinia virus (VACV), used for smallpox vaccination. While smallpox has been eradicated, MPXV and related poxviruses continue to pose a global health threat. Monkeypox (Mpox), similar in clinical presentation to smallpox but milder, is endemic in Central and West Africa. Sporadic outbreaks emphasize the potential for wider dissemination. Understanding their biology, transmission, immune evasion, and clinical features informs disease control strategies. The intersection of medical innovation and biotechnology with poxviruses underscores their importance in both disease and scientific advancement. Further research is essential to enhance prevention, management, and therapeutic interventions for these viruses.
... The adaptive immune system, including T cells and B cells, responds to the infection by producing specific antibodies and cytotoxic T cells. These components are critical for controlling the virus (Saghazadeh and Rezaei 2022). Monkeypox virus causes a range of clinical manifestations in infected individuals. ...
... The adaptive immune system, including T cells and B cells, responds to the infection by producing specific antibodies and cytotoxic T cells. These components are critical for controlling the virus (Saghazadeh and Rezaei 2022). Monkeypox virus causes a range of clinical manifestations in infected individuals. ...
Monkeypox virus, a member of the Orthopoxvirus genus, has emerged as a significant public health concern, primarily within Central and West Africa. This comprehensive review navigates the complex landscape of Monkeypox, delving into its transmission, clinical presentation, diagnosis, treatment, epidemiology, prevention, and ongoing research challenges. The review begins with an exploration of the virus's origins, virology, and mechanisms of transmission. It examines the diverse clinical manifestations of Monkeypox, ranging from mild flu-like symptoms to severe cases with extensive skin lesions, highlighting the importance of early diagnosis and differential diagnosis from similar diseases. Epidemiologically, Monkeypox exhibits sporadic outbreaks in endemic regions, with the risk of international spread, necessitating robust surveillance and preparedness efforts. Global collaboration and vaccination strategies are essential in controlling outbreaks and preventing widespread dissemination. Lastly, the article underscores the significance of ongoing research in understanding the virus's genetic diversity, host factors influencing susceptibility, and the development of effective Monkeypox vaccines and therapies. The need for a One Health approach, recognizing the interconnectedness of human, animal, and environmental health, is paramount in addressing this emerging infectious disease. In conclusion, this review encapsulates the multifaceted nature of Monkeypox and emphasizes the imperative of continued research, global vigilance, and preparedness to safeguard public health against emerging threats in an interconnected world.
... TLR9 acts as a first-line host, recognizing unmethylated CpG dinucleotides present in the virus (Song et al., 2022). The multi-epitope vaccine was therefore subjected to molecular docking studies with TLR-9, which is activated by several poxviruses and induces resistance against VACV, to predict the preferred orientation to form a stable complex (Gorai et al., 2022;Saghazadeh & Rezaei, 2022). It also plays a possible functional role in the molecular mechanism by which TLR9 specifically recognizes CpG-DNA and signals to the intracellular compartment (Ohto et al., 2015). ...
Monkeypox virus (MPV) is closely related to the smallpox virus, and previous data from Africa suggest that the smallpox vaccine (VARV) is at least 85% effective in preventing MPV. No multi-epitope vaccine has yet been developed to prevent MPV infection. In this work, we used in silico structural biology and advanced immunoinformatic strategies to design a multi-epitope subunit vaccine against MPV infection. The designed vaccine sequence is adjuvanted with CpG-ODN and includes HTL/CTL epitopes for similar proteins between vaccinia virus (VACV) that induced T-cell production in vaccinated volunteers and the first draft sequence of the MPV genome associated with the suspected outbreak in several countries, May 2022. In addition, the specific binding of the modified vaccine and the immune Toll-like receptor 9 (TLR9) was estimated by molecular interaction studies. Strong interaction in the binding groove as well as good docking scores confirmed the stringency of the modified vaccine. The stability of the interaction was confirmed by a classical molecular dynamics simulation and normal mode analysis. Then, the immune simulation also indicated the ability of this vaccine to induce an effective immune response against MPV. Codon optimization and in silico cloning of the vaccine into the pET-28a (+) vector also showed its expression potential in the E. coli K12 system. The promising data obtained from the various in silico studies indicate that this vaccine is effective against MPV. However, additional in vitro and in vivo studies are still needed to confirm its efficacy.Communicated by Ramaswamy H. Sarma.
... The genus Orthopoxvirus belongs to the Poxviridae family. Poxviruses, including Monkeypox virus (MPXV), are large, enveloped viruses consisting of double-stranded DNA of approximately 200 Kbp; they have a diameter of up to 400 nm and code for about 190 genes (1). Upon infection, the viral cycle occurs in the cytoplasm of infected cells; unlike most DNA viruses, poxviruses can complete their life cycle within the cell cytoplasm without invading the nucleus (2,3). ...
Introduction
In early to mid-2022, an unexpected outbreak of Monkeypox virus infections occurred outside the African endemic regions. Vaccines originally developed in the past to protect against smallpox are one of the available countermeasures to prevent and protect against Orthopoxvirus infections. To date, there are few studies on the cross-reactivity of neutralizing antibodies elicited by previous vaccinia virus-based vaccination and/or Monkeypox virus infection. The aim of this study was to evaluate a possible approach to performing Monkeypox and vaccinia live-virus microneutralization assays in which the read-out is based on the production of cytopathic effect in the cell monolayer.
Methods
Given the complexity of Orthopoxviruses, the microneutralization assay was performed in such a way as to uncover a potential role of complement, with and without the addition of an external source of Baby Rabbit Complement. A set of human serum samples from individuals who had been naturally infected with Monkeypox virus and individuals who may have and not have undergone vaccinia virus vaccinations, was used to evaluate the performance, sensitivity, and specificity of the assay.
Results and conclusions
The results of the present study confirm the presence and cross-reactivity of antibodies elicited by vaccinia-based vaccines, which proved able to neutralize the Monkeypox virus in the presence of an external source of complement.
... CD11a is to normal lymphocyte development [11]. Some studies confirm that NK cells infected with MPXV are often associated with a Th1 response, NK-cell activation, IFN-γ production, and T-cell activation [12]. TGF-β1 suppresses the functions of Th1 and Th2, NK cells, and CD4 + effector cells and promotes the generation of Treg cells. ...
Introduction
Workers in the healthcare industry form the backbone of health systems everywhere. In the face of global health crises like the current monkeypox (mpox) outbreak, healthcare workers like doctors, dentists, pharmacists, nurses, midwives, paramedics, administrators, support staff, laboratory technicians, and community health workers all play crucial roles in providing care and containing the spread of the disease.
Aim
Therefore, in the wake of concerns about mpox recurrence, we seek to shed light on the occupational transmission of mpox infection and the possible risk to healthcare personnel.
Results
Contamination of the environment of the household of cases of mpox and environment of the patient care units with the viral DNA has been reported besides asymptomatic cases and detection of viral DNA in air samples; therefore, more research on non-lesion-based testing for human mpox infection for screening asymptomatic people, particularly among populations at high risk of infection, in the event of asymptomatic transmission and potential transmission via aerosols is necessary. Monitoring efforts can be aided by incorporating mpox testing into locations where people are more likely to contract illnesses and seek medical attention. We must take a precautionary infection control approach to control the spread of the virus while completing urgent research to understand better the human-to-human mpox transmission process.
Conclusions
In this minireview, we discuss the potential routes of mpox transmission to healthcare and preventative strategies and measures that should be taken and considered.
Graphical Abstract
... Furthermore, the Toll-like receptors (TLRs) are membrane-bound and intracellular receptors that facilitate the identification of damage-and pathogen-associated molecular patterns (DAMPs and PAMPs). A study suggested that innate signaling pathways dependent on TLRs are activated by poxvirus infection (60). TLRs activated or targeted by poxvirus infection have both advantageous and adverse effects on the host. ...
Monkeypox virus (MPXV) is a dsDNA virus, belonging to Poxviridae family. The outbreak of monkeypox disease in humans is critical in European and Western countries, owing to its origin in African regions. The highest number of cases of the disease were found in the United States, followed by Spain and Brazil. Understanding the complete infection mechanism of diverse MPXV strains and their interaction with humans is important for therapeutic drug development, and to avoid any future epidemics. Using computational systems biology, we deciphered the genome-wide protein-protein interactions (PPIs) between 22 MPXV strains and human proteome. Based on phylogenomics and disease severity, 3 different strains of MPXV: Zaire-96-I-16, MPXV-UK_P2, and MPXV_USA_2022_MA001 were selected for comparative functional analysis of the proteins involved in the interactions. On an average, we predicted around 92,880 non-redundant PPIs between human and MPXV proteomes, involving 8014 host and 116 pathogen proteins from the 3 strains. The gene ontology (GO) enrichment analysis revealed 10,624 common GO terms in which the host proteins of 3 strains were highly enriched. These include significant GO terms such as platelet activation (GO:0030168), GABA-A receptor complex (GO:1902711), and metalloendopeptidase activity (GO:0004222). The host proteins were also significantly enriched in calcium signaling pathway (hsa04020), MAPK signaling pathway (hsa04010), and inflammatory mediator regulation of TRP channels (hsa04750). These significantly enriched GO terms and KEGG pathways are known to be implicated in immunomodulatory and therapeutic role in humans during viral infection. The protein hubs analysis revealed that most of the MPXV proteins form hubs with the protein kinases and AGC kinase C-terminal domains. Furthermore, subcellular localization revealed that most of the human proteins were localized in cytoplasm (29.22%) and nucleus (26.79%). A few drugs including Fostamatinib, Tamoxifen and others were identified as potential drug candidates against the monkeypox virus disease. This study reports the genome-scale PPIs elucidation in human-monkeypox virus pathosystem, thus facilitating the research community with functional insights into the monkeypox disease infection mechanism and augment the drug development.
... Innate immune cells, such as dendritic cells, macrophages, and natural killer cells, are activated early in the infection and secrete pro-inflammatory cytokines and chemokines, which recruit additional immune cells to the site of infection. The adaptive immune response to the monkeypox virus involves the production of virus-specific antibodies and T cells, which can recognize and eliminate infected cells [60]. The Poxviridae family virus develops many strategies to escape the host's immune response to infection. ...
Acanthamoeba is well known to host a variety of microorganisms such as viruses, bacteria, protozoa, and yeast. Given the recent number of cases of monkeypox infection, we speculate that amoebae may be aiding viral transmission to the susceptible hosts. Although there is no confirmatory evidence to suggest that Acanthamoeba is a host to monkeypox (a double-stranded DNA virus), the recent discovery of mimivirus (another double-stranded DNA virus) from Acanthamoeba, suggests that amoebae may shelter monkeypox virus. Furthermore, given the possible spread of monkeypox virus from animals to humans during an earlier outbreak, which came about after patients came in contact with prairie dogs, it is likely that animals may also act as mixing vessel between ubiquitously distributed Acanthamoeba and monkeypox virus, in addition to the environmental habitat that acts as an interface in complex interactions between diverse microorganisms and the host.
Monkeypox was caused by Monkeypox Virus (MPXV) and can infect both humans and animals. An understanding of the interplay between host immunity and genetic diversity was necessary to understand the etiology and epidemiology of monkeypox disease. Objective: To clarify how genetic differences and host immune responses interact when a moneypox infection occurs. Furthermore, we also aim to provide insights into individual variability in illness outcomes and possible treatment targets by investigating how distinct genetic profiles affect immune system activation and efficacy. Methods: We have thoroughly analyzed recent research on monkeypox, concentrating on the immune response mechanisms of the host and genetic variables linked to virus vulnerability. For this purpose, the data was searched from various research engines such as google scholar, pubmed, medline etc., by using different key words i.e., monkeypox and host immunity, monkeypox and antibodies interactions, monkeypox outbreak, mnkeypox strains. Conclusions: The way a monkeypox infection progresses and turns out was greatly influenced by the interplay between host genetic differences and immunological responses. Public health initiatives and the creation of tailored treatment plans can both benefit from the identification of genetic markers linked to immunological response profiles and vulnerability.
The recently re-emerged mpox (monkeypox) virus that causes mpox disease is a member of genus Orthopoxvirus and has unprecedentedly spread worldwide. Numerous studies have contributed to our understanding of its evolution, pathophysiology, and clinical manifestations. The current outbreak of the mpox virus depicts its novel route of transmission as a new variant. However, the exact reason for its transition from an epidemic to a pandemic remains unclear. Furthermore, other poxviruses such as vaccinia virus, variola virus, and cowpox virus, also belong to the same genus, Orthopoxvirus . In the present review, our objective was to summarize the evidence on evolution, pathophysiology, and clinical manifestations of mpox virus and its related poxviruses. The present review would aid in a better understanding of the current circulating mpox virus and its differences from other poxviruses. In addition, the shared genetic factors contributing to virulence in these Orthopoxvirus highlight their evolutionary connections and genetic similarities. While they exhibit differences in virulence, studying these genetic relationships is crucial for understanding their biology, pathogenicity, and the development of effective vaccines and antiviral therapeutics to curb mpox disease.
Monkeypox virus, a close relative of variola virus, has significantly increased the incidence of monkeypox disease in humans, with several clinical symptoms. The sporadic spread of the disease outbreaks has resulted in the need for a comprehensive understanding of the molecular mechanisms underlying disease infection and potential therapeutic targets. Protein–protein interactions play a crucial role in various cellular processes and regulate different immune signals during virus infection. Computational algorithms have gained high significance in the prediction of potential protein interaction pairs. Here, we developed a comprehensive database called HuPoxNET (https://kaabil.net/hupoxnet/) using the state-of-the-art MERN stack technology. The database leverages two sequence-based computational models to predict strain-specific protein–protein interactions between human and monkeypox virus proteins. Furthermore, various protein annotations of the human and viral proteins such as gene ontology, KEGG pathways, subcellular localization, protein domains, and novel drug targets identified from our study are also available on the database. HuPoxNET is a user-friendly platform for the scientific community to gain more insights into the monkeypox disease infection and aid in the development of therapeutic drugs against the disease.
Smallpox was a significant cause of mortality for over three thousand years, amounting to 10% of deaths yearly. Edward Jenner discovered smallpox vaccination in 1796, which rapidly became a smallpox infection preventive practice throughout the world and eradicated smallpox infection by 1980. After smallpox eradication, monkeypox vaccines have been used primarily in research and in outbreaks in Africa, where the disease is endemic. In the present, the vaccines are being used for people who work with animals or in high-risk areas, as well as for healthcare workers treating patients with monkeypox. Among all orthopoxviruses (OPXV), monkeypox viral (MPXV) infection occurs mainly in cynomolgus monkeys, natural reservoirs, and occasionally causes severe multi-organ infection in humans, who were the incidental hosts. The first case of the present epidemic of MXPV was identified on May 7, 2022, and rapidly increased the number of cases. In this regard, the WHO declared the outbreak, an international public health emergency on July 23, 2022. The first monkeypox vaccine was developed in the 1960s by the US Army and was based on the vaccinia virus, which is also used in smallpox vaccines. In recent years, newer monkeypox vaccines have been developed based on other viruses such as Modified Vaccinia Ankara (MVA). These newer vaccines are safer and can provide longer-lasting immunity with fewer side effects. For the future, there is ongoing research to improve the current vaccines and to develop new ones. One notable advance has been the development of a recombinant vaccine that uses a genetically modified vaccinia virus to express monkeypox antigens. This vaccine has shown promising results in pre-clinical trials and is currently undergoing further testing in clinical trials. Another recent development has been the use of a DNA vaccine, which delivers genetic material encoding monkeypox antigens directly into cells. This type of vaccine has shown effectiveness in animal studies and is also undergoing clinical testing in humans. Overall, these recent advances in monkeypox vaccine development hold promise for protecting individuals against this potentially serious disease.
In 2022, just before the COVID-19 pandemic ended, many countries noticed a viral monkeypox outbreak. Monkeypox virus, a zoonotic pathogen, causes a febrile illness in humans and resembles smallpox. Prevention strategies encompass vaccination, strict infection control measures, and avoiding contact with infected persons. As monkeypox and related poxviruses continue to pose challenges, ongoing surveillance, early diagnosis, prompt isolation, and effective control measures are crucial for limiting transmission and mitigating the impact of outbreaks on public health. This review provides valuable insights into the evolution of the monkeypox virus and its various modes of transmission, including postmortem transmission, and offers an overall perspective on the guidelines issued by the Government of India to prevent and effectively control the spread of this disease.
In the realm of gene therapy, a pivotal moment arrived with Paul Berg’s groundbreaking identification of the first recombinant DNA in 1972. This achievement set the stage for future breakthroughs. Conditions once considered undefeatable, like melanoma, pancreatic cancer, and a host of other ailments, are now being addressed at their root cause—the genetic level. Presently, the gene therapy landscape stands adorned with 22 approved in vivo and ex vivo products, including IMLYGIC, LUXTURNA, Zolgensma, Spinraza, Patisiran, and many more. In this comprehensive exploration, we delve into a rich assortment of 16 drugs, from siRNA, miRNA, and CRISPR/Cas9 to DNA aptamers and TRAIL/APO2L, as well as 46 carriers, from AAV, AdV, LNPs, and exosomes to naked mRNA, sonoporation, and magnetofection. The article also discusses the advantages and disadvantages of each product and vector type, as well as the current challenges faced in the practical use of gene therapy and its future potential.
As of 29 August 2023, a total of 89,596 confirmed cases of Mpox (monkeypox) have been documented across 114 countries worldwide, with 157 reported fatalities. The Mpox outbreak that transpired in 2022 predominantly affected young men who have sex with men (MSM). While most cases exhibited a mild clinical course, individuals with compromised immune systems, particularly those living with HIV infection and possessing a CD4 count below 200 cells/mm3, experienced a more severe clinical trajectory marked by heightened morbidity and mortality. The approach to managing Mpox is primarily symptomatic and supportive. However, in instances characterized by severe or complicated manifestations, the utilization of antiviral medications becomes necessary. Despite tecovirimat’s lack of official approval by the FDA for treating Mpox in humans, a wealth of positive clinical experiences exists, pending the outcomes of ongoing clinical trials. Brincidofovir and cidofovir have also been administered in select cases due to the unavailability of tecovirimat. Within the scope of this narrative review, our objective was to delve into the clinical attributes of Mpox and explore observational studies that shed light on the utilization of these antiviral agents.
Bioindicators of wildlife health are useful tools for studying the viability of various organisms and populations, and can include a range of phenotypic variables, such as behavior, body size, and physiological parameters, such as circulating hormones and nutrients. Few studies have investigated the utility of total plasma protein as a predictor of environmental or nutritional variation among birds, as well as variation across different habitats and life-history stages. Here, we examined relationships between plasma protein and season, urbanization, sex, body condition, molt status, and disease state in House Finches (Haemorhous mexicanus). We sampled blood from House Finches across three seasons (winter, summer, and fall 2021) and measured plasma protein levels using a Bradford assay. We also collected data including condition, sex, and poxvirus infection state at capture, as well as fecal samples to assess gut parasitism (coccidiosis). During the fall season, we also estimated molt status, as number of actively growing feathers. We found a significant relationship between circulating protein levels and capture site, as well as novel links to molt state and pox presence, with urban birds, those infected with pox, and those in more intense molt having higher protein levels. Our results support the hypotheses that plasma protein concentration can be indicative of a bird’s body molt and degree of habitat urbanization, although future work is needed to determine why protein levels were higher in virus-infected birds.
A healthy young man first diagnosed with mpox in May 2022 presented again in November 2022 with anal proctitis and a positive PCR on a rectal swab for MPX virus (MPXV) after a recent trip to Brazil, where he engaged in condomless sexual intercourse with multiple male partners.
Inborn errors of immunity (IEI) are disorders characterized by the impaired development and functioning of the immune system components. These errors can affect cellular immunity, humoral immunity, or both, predisposing patients to localized and systemic manifestations (for the most recent update on the classification of IEI, see [1](#ref‐0001)). Among various signs and symptoms, increased susceptibility to infections and autoimmune and inflammatory conditions with a high tendency to involve the skin, lungs, and gut are most‐documented. With the re‐emergence of the monkeypox (MPX) outbreak in 2022 and a new picture that it has presented, i.e., the global spread of the disease and the high prevalence in patients with human immunodeficiency syndrome (HIV) [2](#ref‐0002), worries are increasing about the possible threat of monkeypox virus (MPXV) to affect patients with IEI. As of November 04, 2022, 109 countries reported a total of more than 78,000 MPXV‐infected cases, with a death rate of about 0.01% [3](#ref‐0003). During this outbreak, males who have sex with other males (MSM) have constituted the majority of the MPXV‐infected population [4,5](#ref‐0004). Moreover, people with HIV account for about 40% of this population and exhibit a more severe disease course, as represented in the higher need for hospitalization [6](#ref‐0006). To date, no infection with MPXV has been reported in patients with IEI; however, high susceptibility of immunocompromised people to this infection is alarming for people with IEI and their caregivers. Below address a few important questions concerning patients with IEI during this outbreak. This article is protected by copyright. All rights reserved.
An immunocompromised status has been associated with more odds of being infected with monkeypox virus (MPXV) and progressing to severe disease. This aligns with the importance of immune competence for MPXV control and clearance. We and others have previously reviewed parallels between MPXV and other viruses belonging to the Poxviridae in affecting the immune system. This article reviews studies providing direct evidence of the MPXV-immune interactions. The wide-ranging effects of MPXV on the immune system, from stimulation to modulation to memory, are broadly categorised, followed by a detailing of these effects on the immune cells and molecules, including natural killer cells, macrophages, neutrophils, lymphocytes, cytokines, interferons, chemokines, and complement.
Modified vaccinia Ankara (MVA) is an attenuated strain of vaccinia virus (VACV), a dsDNA virus that replicates its genome in the cytoplasm and as a result is canonically sensed by the cyclic GMP-AMP synthase (cGAS) and its downstream stimulator of interferon genes (STING). MVA has a highly restricted host range due to major deletions in its genome including inactivation of immunomodulatory genes, only being able to grow in avian cells and the hamster cell line BHK21. Here we studied the interplay between MVA and the cGAS/STING DNA in this permissive cell line and determined whether manipulation of this axis could impact MVA replication and cell responses. We demonstrate that BHK21 cells retain a functional cGAS/STING axis that responds to canonical DNA sensing agonists, upregulating interferon stimulated genes (ISGs). BHK21 cells also respond to MVA, but with a distinct ISG profile. This profile remains unaltered after CRISPR/Cas9 knock-out editing of STING and ablation of cytosolic DNA responses, indicating that MVA responses are independent of the cGAS/STING axis. Furthermore, infection by MVA diminishes the ability of BHK21 cells to respond to exogenous DNA suggesting that MVA still encodes uncharacterised inhibitors of DNA sensing. This suggests that using attenuated strains in permissive cell lines may assist in identification of novel host-virus interactions that may be of relevance to disease or the therapeutic applications of poxviruses.
The pulmonary immune system consists of a network of tissue-resident cells as well as immune cells that are recruited to the lungs during infection and/or inflammation. How these immune components function during an acute poxvirus infection is not well understood. Intranasal infection of mice with vaccinia virus causes lethal pneumonia and systemic dissemination. Here we report that vaccinia C7 is a crucial virulence factor that blocks activation of the transcription factor IRF3. We provide evidence that type II alveolar epithelial cells (AECIIs) respond to pulmonary infection of vaccinia virus by inducing IFN-β and IFN-stimulated genes via the activation of the MDA5 and STING-mediated nucleic acid-sensing pathways and the type I IFN positive feedback loop. This leads to the recruitment and activation of CCR2+ inflammatory monocytes in the infected lungs and subsequent differentiation into Lyve1− interstitial macrophages (Lyve1− IMs), which efficiently engulf viral particles and block viral replication. Our results provide insights into how innate immune sensing of viral infection by lung AECIIs influences the activation and differentiation of CCR2+ inflammatory monocytes to defend against pulmonary poxvirus infection. Smallpox is a highly contagious respiratory pathogen associated with a high mortality rate. Here the authors utilize a mouse model of intranasal vaccinia virus infection and show a C7 gene encoded virulence factor attenuates type I IFN release by lung type II alveolar epithelial cells and reduces lung inflammatory monocyte responses.
Monkeypox, a zoonotic disease caused by an orthopoxvirus, results in a smallpox-like disease in humans. Since monkeypox in humans was initially diagnosed in 1970 in the Democratic Republic of the Congo (DRC), it has spread to other regions of Africa (primarily West and Central), and cases outside Africa have emerged in recent years. We conducted a systematic review of peer-reviewed and grey literature on how monkeypox epidemiology has evolved, with particular emphasis on the number of confirmed, probable, and/or possible cases, age at presentation, mortality, and geographical spread. The review is registered with PROSPERO (CRD42020208269). We identified 48 peer-reviewed articles and 18 grey literature sources for data extraction. The number of human monkeypox cases has been on the rise since the 1970s, with the most dramatic increases occurring in the DRC. The median age at presentation has increased from 4 (1970s) to 21 years (2010–2019). There was an overall case fatality rate of 8.7%, with a significant difference between clades—Central African 10.6% (95% CI: 8.4%– 13.3%) vs. West African 3.6% (95% CI: 1.7%– 6.8%). Since 2003, import- and travel-related spread outside of Africa has occasionally resulted in outbreaks. Interactions/activities with infected animals or individuals are risk behaviors associated with acquiring monkeypox. Our review shows an escalation of monkeypox cases, especially in the highly endemic DRC, a spread to other countries, and a growing median age from young children to young adults. These findings may be related to the cessation of smallpox vaccination, which provided some cross-protection against monkeypox, leading to increased human-to-human transmission. The appearance of outbreaks beyond Africa highlights the global relevance of the disease. Increased surveillance and detection of monkeypox cases are essential tools for understanding the continuously changing epidemiology of this resurging disease.
Poxviridae have developed a plethora of strategies to evade innate and adaptive immunity. In this review, we focused on the vaccinia virus E3 protein, encoded by the E3L gene. E3 is present within the Chordopoxvirinae subfamily (with the exception of the avipoxviruses and molluscum contagiosum virus) and displays pleiotropic effects on the innate immune system. Initial studies identified E3 as a double-stranded RNA (dsRNA)-binding protein (through its C terminus), able to inhibit the activation of protein kinase dependent on RNA (PKR) and the 2′5′-oligoadenylate synthetase (OAS)/RNase L pathway, rendering E3 a protein counteracting the type I interferon (IFN) system. In recent years, N-terminal mutants of E3 unable to bind to Z-form nucleic acids have been shown to induce the cellular death pathway necroptosis. This pathway was dependent on host IFN-inducible Z-DNA-binding protein 1 (ZBP1); full-length E3 is able to inhibit ZBP1-mediated necroptosis. Binding to what was identified as Z-RNA has emerged as a novel mechanism of counteracting the type I IFN system and has broadened our understanding of innate immunity against viral infections. This article gives an overview of the studies leading to our understanding of the vaccinia virus E3 protein function and its involvement in viral pathogenesis. Furthermore, a short summary of other viral systems is provided.
Regulated cell death frequently occurs upon infection by intracellular pathogens, and extent and regulation is often cell-type-specific. We aimed to identify the cell death-signaling pathways triggered in macrophages by infection with modified vaccinia virus Ankara (MVA), an attenuated strain of vaccinia virus used in vaccination. While most target cells seem to be protected by antiapoptotic proteins encoded in the MVA genome, macrophages die when infected with MVA. We targeted key signaling components of specific cell death-pathways and pattern recognition-pathways using genome editing and small molecule inhibitors in an in vitro murine macrophage differentiation model. Upon infection with MVA, we observed activation of mitochondrial and death-receptor-induced apoptosis-pathways as well as the necroptosis-pathway. Inhibition of individual pathways had a little protective effect but led to compensatory death through the other pathways. In the absence of mitochondrial apoptosis, autocrine/paracrine TNF-mediated apoptosis and, in the absence of caspase-activity, necroptosis occurred. TNF-induction depended on the signaling molecule STING, and MAVS and ZBP1 contributed to MVA-induced apoptosis. The mode of cell death had a substantial impact on the cytokine response of infected cells, indicating that the immunogenicity of a virus may depend not only on its PAMPs but also on its ability to modulate individual modalities of cell death. These findings provide insights into the diversity of cell death-pathways that an infection can trigger in professional immune cells and advance our understanding of the intracellular mechanisms that govern the immune response to a virus.
Host pattern recognition receptors (PRRs) sense pathogen-associated molecular patterns (PAMPs), which are molecular signatures shared by different pathogens. Recognition of PAMPs by PRRs initiate innate immune responses via diverse signaling pathways. Over recent decades, advances in our knowledge of innate immune sensing have enhanced our understanding of the host immune response to poxviruses. Multiple PRR families have been implicated in poxvirus detection, mediating the initiation of signaling cascades, activation of transcription factors, and, ultimately, the expression of antiviral effectors. To counteract the host immune defense, poxviruses have evolved a variety of immunomodulators that have diverse strategies to disrupt or circumvent host antiviral responses triggered by PRRs. These interactions influence the outcomes of poxvirus infections. This review focuses on our current knowledge of the roles of PRRs in the recognition of poxviruses, their elicited antiviral effector functions, and how poxviral immunomodulators antagonize PRR-mediated host immune responses.
Sheeppox (SPP) is a highly contagious disease of small ruminants caused by sheeppox virus (SPPV) and predominantly occurs in Asia and Africa with significant economic losses. SPPV is genetically and immunologically closely related to goatpox virus (GTPV) and lumpy skin disease virus (LSDV), which infect goats and cattle respectively. SPPV live attenuated vaccines (LAVs) are used for vaccination against SPP and goatpox (GTP). Mechanisms related to innate immunity elicited by SPPV are unknown. Although adaptive immunity is responsible for long-term immunity, it is the innate responses that prevent viral invasion and replication before LAVs generate specific long-term protection. We analyzed the relative expression of thirteen selected genes that included pattern recognition receptors (PRRs), Nuclear factor-κβ p65 (NF-κβ), and cytokines to understand better the interaction between SPPV and its host. The transcripts of targeted genes in sheep PBMC incubated with either wild type (WT) or LAV SPPV were analyzed using quantitative PCR. Among PRRs, we observed a significantly higher expression of RIG-1 in PBMC incubated with both WT and LAV, with the former producing the highest expression level. However, there was high inter-individual variability in cytokine transcripts levels among different donors, with the expression of TNFα, IL-15, and IL-10 all significantly higher in both PBMC infected with either WT or LAV compared to control PBMC. Correlation studies revealed a strong significant correlation between RIG-1 and IL-10, between TLR4, TNFα, and NF-κβ, between IL-18 and IL-15, and between NF-κβ and IL-10. There was also a significant negative correlation between RIG-1 and IFNγ, between TLR3 and IL-1 β, and between TLR4 and IL-15 (P< 0.05). This study identified RIG-1 as an important PRR in the signaling pathway of innate immune activation during SPPV infection, possibly through intermediate viral dsRNA. The role of immunomodulatory molecules produced by SPPV capable of inhibiting downstream signaling activation following RIG-1 upregulation is discussed. These findings advance our knowledge of the induction of immune responses by SPPV and will help develop safer and more potent vaccines against SPP and GTP.
Poxviruses comprise many members that infect both vertebrate and invertebrate animals, including humans. Despite the eradication of the historically notorious smallpox, poxviruses remain significant public health concerns and serious endemic diseases. This short review briefly summarizes the present, historical, and future threats posed by poxviruses to public health, wildlife and domestic animals, the role poxviruses have played in shaping modern medicine and biomedical sciences, the insight poxviruses have provided into complex life processes, and the utility of poxviruses in biotechniques and in fighting other infectious diseases and cancers. It is anticipated that readers will appreciate the great merit and need for continued strong support of poxvirus research; research which benefits not only the expansion of fundamental biological knowledge but also the battle against diverse diseases.
Type I interferons (IFN-I) are antiviral cytokines that signal through the ubiquitous IFN-I receptor (IFNAR). Following footpad infection with ectromelia virus (ECTV), a mouse-specific pathogen, C57BL/6 (B6) mice survive without disease, while B6 mice broadly deficient in IFNAR succumb rapidly. We now show that for survival to ECTV, only hematopoietic cells require IFNAR expression. Survival to ECTV specifically requires IFNAR in both natural killer (NK) cells and monocytes. However, intrinsic IFNAR signaling is not essential for adaptive immune cell responses or to directly protect non-hematopoietic cells such as hepatocytes, which are principal ECTV targets. Mechanistically, IFNAR-deficient NK cells have reduced cytolytic function, while lack of IFNAR in monocytes dampens IFN-I production and hastens virus dissemination. Thus, during a pathogenic viral infection, IFN-I coordinates innate immunity by stimulating monocytes in a positive feedback loop and by inducing NK cell cytolytic function.
RNA helicase A/DHX9 is required for diverse RNA-related essential cellular functions, anti-viral responses and also hijacked by RNA viruses to support their replication. Here, we show that during the late replication stage in human cancer cells of myxoma virus (MYXV), a member of the dsDNA poxvirus family that is being developed as an oncolytic virus, DHX9 forms unique granular cytoplasmic structures, which we named “DHX9 anti-viral granules”. These DHX9 anti-viral granules are not formed if MYXV DNA replication and/or late protein synthesis is blocked. When formed, DHX9 anti-viral granules significantly reduced nascent protein synthesis in the MYXV-infected cancer cells. MYXV late gene(s) transcription and translation was also significantly compromised, particularly in non-permissive or semi-permissive human cancer cells where MYXV replication is partly or completely restricted. Directed knockdown of DHX9 significantly enhanced viral late protein synthesis and progeny virus formation in normally restrictive cancer cells. We further demonstrate that DHX9 is not a component of the canonical cellular stress granules. DHX9 anti-viral granules are induced by MYXV, and other poxviruses, in human cells, and are associated with other known cellular components of stress granules, dsRNA and virus encoded dsRNA-binding protein M029, a known interactor with DHX9. Thus, DHX9 anti-viral granules function by hijacking poxviral elements needed for the cytoplasmic viral replication factories. These results demonstrate a novel anti-viral function for DHX9 that is recruited from the nucleus into the cytoplasm and this step can be exploited to enhance oncolytic virotherapy against the subset of human cancer cells that normally restrict MYXV.
Importance
The cellular DHX9 has both pro-viral and anti-viral roles against diverse RNA and DNA viruses. In this manuscript we demonstrate that DHX9 can form unique anti-viral granules in the cytoplasm during MYXV replication in human cancer cells. These anti-viral granules sequester viral proteins and reduce viral late protein synthesis and thus regulate MYXV, and other poxviruses, that replicate in the cytoplasm. In addition, we show that in the absence of DHX9 the formation of DHX9 anti-viral granules can be inhibited which significantly enhanced oncolytic MYXV replication in human cancer cell lines where the virus is normally restricted. Our results also show that DHX9 anti-viral granules are formed after viral infection but not by common nonviral cellular stress inducers. Thus, our study suggests that DHX9 has anti-viral activity in human cancer cells and this pathway can be targeted for enhanced activity of oncolytic poxviruses against even restrictive cancer cells.
The anti-viral immune response is dependent on the ability of infected cells to sense foreign nucleic acids. In multiple species, the pattern recognition receptor (PRR) cyclic GMP-AMP synthase (cGAS) senses viral DNA as an essential component of the innate response. cGAS initiates a range of signaling outputs that are dependent on generation of the second messenger cGAMP that binds to the adaptor protein stimulator of interferon genes (STING). Here we show that in chicken macrophages, the cGAS/STING pathway is essential not only for the production of type-I interferons in response to intracellular DNA stimulation, but also for regulation of macrophage effector functions including the expression of MHC-II and co-stimulatory molecules. In the context of fowlpox, an avian DNA virus infection, the cGAS/STING pathway was found to be responsible for type-I interferon production and MHC-II transcription. The sensing of fowlpox virus DNA is therefore essential for mounting an anti-viral response in chicken cells and for regulation of a specific set of macrophage effector functions.
Poxvirus systems have been extensively used as vaccine vectors. Herein a RNA-Seq analysis of intramuscular injection sites provided detailed insights into host innate immune responses, as well as expression of vector and recombinant immunogen genes, after vaccination with a new multiplication defective, vaccinia-based vector, Sementis Copenhagen Vector. Chikungunya and Zika virus immunogen mRNA and protein expression was associated with necrosing skeletal muscle cells surrounded by mixed cellular infiltrates. The multiple adjuvant signatures at 12 hours post-vaccination were dominated by TLR3, 4 and 9, STING, MAVS, PKR and the inflammasome. Th1 cytokine signatures were dominated by IFNγ, TNF and IL1β, and chemokine signatures by CCL5 and CXCL12. Multiple signatures associated with dendritic cell stimulation were evident. By day seven, vaccine transcripts were absent, and cell death, neutrophil, macrophage and inflammation annotations had abated. No compelling arthritis signatures were identified. Such injection site vaccinology approaches should inform refinements in poxvirus-based vector design.
Complex dermal wounds represent major medical and financial burdens, especially in the context of comorbidities such as diabetes, infection and advanced age. New approaches to accelerate and improve, or "fine tune" the healing process, so as to improve the quality of cutaneous wound healing and management, are the focus of intense investigation. Here, we investigate the topical application of a recombinant immune modulating protein which inhibits the interactions of chemokines with glycosaminoglycans, reducing damaging or excess inflammation responses in a splinted full-thickness excisional wound model in mice. M-T7 is a 37 kDa-secreted, virus-derived glycoprotein that has demonstrated therapeutic efficacy in numerous animal models of inflammatory immunopathology. Topical treatment with recombinant M-T7 significantly accelerated wound healing when compared to saline treatment alone. Healed wounds exhibited properties of improved tissue remodeling, as determined by collagen maturation. M-T7 treatment accelerated the rate of peri-wound angiogenesis in the healing wounds with increased levels of TNF, VEGF and CD31. The immune cell response after M-T7 treatment was associated with a retention of CCL2 levels, and increased abundances of arginase-1-expressing M2 macrophages and CD4 T cells. Thus, topical treatment with recombinant M-T7 promotes a pro-resolution environment in healing wounds, and has potential as a novel treatment approach for cutaneous tissue repair.
Spinal cord injury (SCI) initiates a severe, destructive inflammation with pro-inflammatory, CD68+/CD163−, phagocytic macrophages infiltrating the area of necrosis and hemorrhage by day 3 and persisting for the next 16 weeks. Inhibition of macrophage infiltration of the site of necrosis that is converted into a cavity of injury (COI) during the first week post-SCI, should limit inflammatory damage, shorten its duration and result in neuroprotection. By sustained subdural infusion we administered Serp-1, a Myxoma virus-derived immunomodulatory protein previously shown to improve neurologic deficits and inhibit macrophage infiltration in the COI in rats with the balloon crush SCI. Firstly, in a 7 day long study, we determined that the optimal dose for macrophage inhibition was 0.2 mg/week. Then, we demonstrated that a continuous subdural infusion of Serp-1 for 8 weeks resulted in consistently accelerated lowering of pro-inflammatory macrophages in the COI and in their almost complete elimination similar to that previously observed at 16 weeks in untreated SCI rats. The macrophage count in the COI is a quantitative test directly related to the severity of destructive inflammation initiated by the SCI. This test has consistently demonstrated anti-inflammatory effect of Serp-1 interpreted as neuroprotection, the first and necessary step in a therapeutic strategy in neurotrauma.
Cells contain numerous immune sensors to detect virus infection. The cyclic GMP-AMP (cGAMP) synthase (cGAS) recognizes cytosolic DNA and activates innate immune responses via stimulator of interferon genes (STING), but the impact of DNA sensing pathways on host protective responses has not been fully defined. We demonstrate that cGAS/STING activation is required to resist lethal poxvirus infection. We identified viral Schlafen (vSlfn) as the main STING inhibitor, and ectromelia virus was severely attenuated in the absence of vSlfn. Both vSlfn-mediated virulence and STING inhibitory activity were mapped to the recently discovered poxin cGAMP nuclease domain. Animals were protected from subcutaneous, respiratory, and intravenous infection in the absence of vSlfn, and interferon was the main antiviral protective mechanism controlled by the DNA sensing pathway. Our findings support the idea that manipulation of DNA sensing is an efficient therapeutic strategy in diseases triggered by viral infection or tissue damage-mediated release of self-DNA.
As pattern recognition receptors, cytosolic DNA sensors quickly induce an effective innate immune response. Poxvirus, a large DNA virus, is capable of evading the host antiviral innate immune response. In this review, we summarize the latest studies on how poxvirus is sensed by the host innate immune system and how poxvirus-encoded proteins antagonize DNA sensors. A comprehensive understanding of the interplay between poxvirus and DNA-sensing antiviral immune responses of the host will contribute to the development of new antiviral therapies and vaccines in the future.
The complement system is a key component of innate immunity which readily responds to invading microorganisms. Activation of the complement system typically occurs via three main pathways and can induce various antimicrobial effects, including: neutralization of pathogens, regulation of inflammatory responses, promotion of chemotaxis, and enhancement of the adaptive immune response. These can be vital host responses to protect against acute, chronic, and recurrent viral infections. Consequently, many viruses (including dengue virus, West Nile virus and Nipah virus) have evolved mechanisms for evasion or dysregulation of the complement system to enhance viral infectivity and even exacerbate disease symptoms. The complement system has multifaceted roles in both innate and adaptive immunity, with both intracellular and extracellular functions, that can be relevant to all stages of viral infection. A better understanding of this virus-host interplay and its contribution to pathogenesis has previously led to: the identification of genetic factors which influence viral infection and disease outcome, the development of novel antivirals, and the production of safer, more effective vaccines. This review will discuss the antiviral effects of the complement system against numerous viruses, the mechanisms employed by these viruses to then evade or manipulate this system, and how these interactions have informed vaccine/therapeutic development. Where relevant, conflicting findings and current research gaps are highlighted to aid future developments in virology and immunology, with potential applications to the current COVID-19 pandemic.
The wild-derived inbred CAST/EiJ mouse, one of eight founder strains in the Collaborative Cross panel, is an exceptional model for studying monkeypox virus (MPXV), an emerging human pathogen, and other orthopoxviruses including vaccinia virus (VACV). Previous studies suggested that the extreme susceptibility of the CAST mouse to orthopoxviruses is due to an insufficient innate immune response. Here, we focused on the low number of natural killer (NK) cells in the naïve CAST mouse as a contributing factor to this condition. Administration of IL-15 to CAST mice transiently increased NK and CD8⁺ T cells that could express IFN-γ, indicating that the progenitor cells were capable of responding to cytokines. However, the number of NK cells rapidly declined indicating a defect in their homeostasis. Furthermore, IL-15-treated mice were protected from an otherwise lethal challenge with VACV or MPXV. IL-15 decreased virus spread and delayed death even when CD4⁺/CD8⁺ T cells were depleted with antibody, supporting an early protective role of the expanded NK cells. Purified splenic NK cells from CAST mice proliferated in vitro in response to IL-15 and could be activated with IL-12/IL-18 to secrete interferon-γ. Passive transfer of non-activated or activated CAST NK cells reduced VACV spread but only the latter completely prevented death at the virus dose used. Moreover, antibodies to interferon-γ abrogated the protection by activated NK cells. Thus, the inherent susceptibility of CAST mice to orthopoxviruses can be explained by a low level of NK cells and this vulnerability can be overcome either by expanding their NK cells in vivo with IL-15 or by passive transfer of purified NK cells that were expanded and activated in vitro.
Toll-like receptors (TLRs) are pattern recognition receptors that allow innate immunity to protect our body against invading pathogens. They are also regulators of adaptive immunity. The human TLR was discovered quite recently, but its functional significance is known worldwide and today TLR agonists have been approved for use in humans. This book provides an overview of TLRs and their role in parasitic infections and neurodegenerative diseases. It is hoped that it will encourage readers to seek out the latest developments in TLRs.
Cells sensing infection produce Type I interferons (IFN-I) to stimulate Interferon Stimulated Genes (ISGs) that confer resistance to viruses. During lympho-hematogenous spread of the mouse pathogen ectromelia virus (ECTV), the adaptor STING and the transcription factor IRF7 are required for IFN-I and ISG induction and resistance to ECTV. However, it is unknown which cells sense ECTV and which pathogen recognition receptor (PRR) upstream of STING is required for IFN-I and ISG induction. We found that cyclic-GMP-AMP (cGAMP) synthase (cGAS), a DNA-sensing PRR, is required in bone marrow-derived (BMD) but not in other cells for IFN-I and ISG induction and for resistance to lethal mousepox. Also, local administration of cGAMP, the product of cGAS that activates STING, rescues cGAS but not IRF7 or IFN-I receptor deficient mice from mousepox. Thus, sensing of infection by BMD cells via cGAS and IRF7 is critical for resistance to a lethal viral disease in a natural host.
Infection of adult mice with the clone 13 (CL13) strain of lymphocytic choriomeningitis virus (LCMV) is extensively used as a model of chronic infection. In this paper, we show that mice chronically infected with CL13 succumb to challenge with ectromelia virus (ECTV; the agent of mousepox) and that natural killer (NK) cells in CL13-infected mice are reduced in numbers and have an immature and partially activated phenotype but do respond to ECTV. These data may provide additional clues why humans chronically infected with certain pathogens are less resistant to viral diseases.
The increasingly recognized role of different types of B cells and plasma cells in protective and pathogenic immune responses combined with technological advances have generated a plethora of information regarding the heterogeneity of this human immune compartment. Unfortunately, the lack of a consistent classification of human B cells also creates significant imprecision on the adjudication of different phenotypes to well-defined populations. Additional confusion in the field stems from: the use of non-discriminatory, overlapping markers to define some populations, the extrapolation of mouse concepts to humans, and the assignation of functional significance to populations often defined by insufficient surface markers. In this review, we shall discuss the current understanding of human B cell heterogeneity and define major parental populations and associated subsets while discussing their functional significance. We shall also identify current challenges and opportunities. It stands to reason that a unified approach will not only permit comparison of separate studies but also improve our ability to define deviations from normative values and to create a clean framework for the identification, functional significance, and disease association with new populations.
Poxviruses are large, double-stranded DNA viruses that replicate entirely in the cytoplasm, an unusual act that activates pathogen sensors and innate antiviral responses. In order to replicate, poxviruses therefore encode a wide range of innate immune antagonists that include F17, a protein that dysregulates the kinase mammalian target of rapamycin (mTOR) to suppress interferon-stimulated gene (ISG) responses. However, the host sensor(s) that detects infection in the absence of F17 and its precise contribution to infection remains unknown. Here, we show that the cytosolic DNA sensor cGAS is primarily responsible for activating ISG responses in biologically relevant cell types infected with a poxvirus that does not express F17. However, in line with their expression of ∼100 proteins that act as immune response and ISG antagonists, while F17 helps suppress cGAS-mediated responses, we find that a critical function of its mTOR dysregulation activity is to enhance poxvirus protein production.
Vaccinia virus (VACV) has numerous immune evasion strategies, including multiple mechanisms of inhibition of interferon regulatory factor 3 (IRF-3), nuclear factor kB (NF-kB), and type I interferon (IFN) signaling. Here, we use highly multiplexed proteomics
to quantify ~9,000 cellular proteins and ~80% of viral proteins at seven time points
throughout VACV infection. A total of 265 cellular proteins are downregulated >2-fold by VACV, including putative natural killer cell ligands and IFN-stimulated genes. Two-thirds of these viral targets, including class II histone deacetylase 5 (HDAC5), are degraded proteolytically during infection. In follow-up analysis, we demonstrate that HDAC5 restricts replication of both VACV and herpes simplex virus type 1. By generating a proteinbased temporal classification of VACV gene expression, we identify protein C6, a multifunctional IFN antagonist, as being necessary and sufficient for proteasomal degradation of HDAC5. Our approach thus identifies both a host antiviral factor and a viral
mechanism of innate immune evasion.
Spinal cord injury (SCI)-initiated inflammation was treated with anti-inflammatory reagents. We compared local spinal cord or intraperitoneal infusion of two Myxoma virus derived immune modulating proteins, Serp-1 and M-T7, with dexamethasone (DEX).
Hemorrhage and necrosis after SCI initiate a complex pathogenesis dominated by early, severe and highly destructive inflammatory macrophage infiltration. We examined sustained, 7-day, subdural infusion of either M-T7, a chemokine modulator or Serp-1, a plasminogen activator and factor inhibitor. Mature male rats had epidural balloon crush SCI and sustained subdural infusion of Serp-1, M-T7, DEX or saline for 7 days via the osmotic pump. A separate group of rats with SCI had intra-peritoneal infusion. Clinical evaluation included endpoint monitoring with body weight, hemorrhagic cystitis and bilateral toe pinch response. Sections of the spinal cord were analyzed histologically and macrophage numbers counted by standardized protocol in the cavity of injury (COI).
While the rats administered DEX demonstrated substantial body weight loss, dehydration and dermal atrophy consistent with steroid toxicity, rats infused with Serp-1 and M-T7 had no toxicity. Serp-1 improved withdrawal responses. Subdural infusion of Serp-1, M-T7 and DEX significantly reduced numbers of phagocytic, CD68-positive macrophages. With intraperitoneal infusion only M-T7 reduced macrophage counts, Serp-1 showed only a trend. Local infusion of highly active immune modulating proteins; Serp-1 and M-T7, targeting serine protease and chemokine pathways demonstrated excellent potential for neuroprotection after severe SCI in a rat model, without adverse side effects. Sustained subdural infusion offers an alternative route of administration for treatment of SCI.
Among the repertoire of immunoregulatory proteins encoded by myxoma virus, M013 is a viral homologue of the PYRIN domain-Only Protein (vPOP) family. In myeloid cells, M013 protein has been shown to inhibit both the inflammasome and NF-κB signaling pathways by direct binding to ASC1 and NF-κB1, respectively. In this study, a three-dimensional homology model of the M013 pyrin domain (PYD) was built based on similarities to known PYD structures. A distinctive feature of the deduced surface electrostatic map of the M013 PYD is the presence of a negatively region consisting of numerous aspartate and glutamate residues in close proximity. Single-site mutations of aspartate and glutamate residues reveal their role in interactions with ASC-1. The biological significance of charge complementarity in the M013:ASC-1 interaction was further confirmed by functional assays of caspase-1 activation and subsequent secretion of cytokines. M013 also has a unique 33-residue C-terminal tail that follows the N-terminal PYD, and it is enriched in positively charged residues. Deletion of the tail of M013 significantly inhibited the interactions between M013 and NF-κB1, thus compromising the ability of the viral protein to suppress the secretion of proinflammatory cytokines. These results demonstrate that vPOP M013 exploits distinct structural motifs to regulate both the inflammasome and NF-κB pathways.
B cells expressing the transcription factor T‐bet have emerged as participants in a number of protective and pathogenic immune responses. T‐bet+ B cells characteristically differentiate in response to combined Toll‐like receptor and cytokine signaling, contribute to protective immunity against intracellular pathogens via IgG2a/c production and antibody‐independent mechanisms, and are prone to produce autoantibodies. Despite recent advances, a number of questions remain regarding the basic biology of T‐bet+ B cells and their functional niche within the immune system. Herein, we review the discovery and defining characteristics of the T‐bet+ B cell subset in both mice and humans. We further discuss their origins, the basis for their persistence, and their potential fate in vivo. Evidence indicates that T‐bet+ B cells represent a distinct, germinal center‐derived memory population that may serve as an important therapeutic target for the improvement of humoral immunity and prevention of autoimmunity.
CD40 triggering may result in antitumor effects of potentially high clinical relevance. To gain insights important for patient selection and to identify adequate targeting techniques, we investigated CD40 expression in human cancer tissues and generated a replication-incompetent recombinant vaccinia virus expressing CD40 ligand (rVV40L). Its effects were explored in vitro and in vivo upon direct CD40 targeting on malignant cells or macrophage activation. CD40 expression was analyzed by immunohistochemistry in tumor and stromal cells in a multi-tumor array including 836 specimens from 27 different tumor types. Established tumor cell lines were used to explore the capacity of rVV40L to induce malignant cell apoptosis and modulate functional profiles of polarized macrophages. CD40 expression was detectable in significantly higher numbers of stromal as compared to malignant cells in lung and breast cancers. CD40 ligation following rVV40L infection induced apoptosis in CD40(+) cancer cells, but only in the presence of intact specific signal transduction chain. Importantly, rVV40L infection promoted the induction of TNF-α-dependent antitumor activity of M1-like macrophages directed against CD40(-) targets. CD40-activated M1-like macrophages also displayed enhanced ability to CXCL10-dependently recruit CD8+ T cells and to efficiently present cancer cell intracellular antigens through cross-priming. Moreover, rVV-driven CD40L expression partially “re-educated” M2-like macrophages, as suggested by detectable CXCL10 and IL-12 production. Most importantly, we observed that intra-tumoral injection of rVV40L-infected human macrophages inhibits progression of human CD40(-) tumors in vivo. First evidences of anticancer activity of rVV40L strongly encourage further evaluations.
Cytosolic DNA triggers innate immune responses through the activation of cyclic GMP–AMP synthase (cGAS) and production of the cyclic dinucleotide second messenger 2′,3′-cyclic GMP–AMP (cGAMP)1–4. 2′,3′-cGAMP is a potent inducer of immune signalling; however, no intracellular nucleases are known to cleave 2′,3′-cGAMP and prevent the activation of the receptor stimulator of interferon genes (STING)5–7. Here we develop a biochemical screen to analyse 24 mammalian viruses, and identify poxvirus immune nucleases (poxins) as a family of 2′,3′-cGAMP-degrading enzymes. Poxins cleave 2′,3′-cGAMP to restrict STING-dependent signalling and deletion of the poxin gene (B2R) attenuates vaccinia virus replication in vivo. Crystal structures of vaccinia virus poxin in pre- and post-reactive states define the mechanism of selective 2′,3′-cGAMP degradation through metal-independent cleavage of the 3′–5′ bond, converting 2′,3′-cGAMP into linear Gp[2′–5′]Ap[3′]. Poxins are conserved in mammalian poxviruses. In addition, we identify functional poxin homologues in the genomes of moths and butterflies and the baculoviruses that infect these insects. Baculovirus and insect host poxin homologues retain selective 2′,3′-cGAMP degradation activity, suggesting an ancient role for poxins in cGAS–STING regulation. Our results define poxins as a family of 2′,3′-cGAMP-specific nucleases and demonstrate a mechanism for how viruses evade innate immunity.
TRAFs [tumor necrosis factor (TNF) receptor associated factors] are a family of signaling molecules that function downstream of multiple receptor signaling pathways and play a pivotal role in the biology of innate, and adaptive immune cells. Following receptor ligation, TRAFs generally function as adapter proteins to mediate the activation of intracellular signaling cascades. With the exception of TRAF1 that lacks a Ring domain, TRAFs have an E3 ubiquitin ligase activity which also contributes to their ability to activate downstream signaling pathways. TRAF-mediated signaling pathways culminate in the activation of several transcription factors, including nuclear factor-κB (NF-κB), mitogen-activated protein kinases (MAPKs; e.g., ERK-1 and ERK-2, JNK, and p38), and interferon-regulatory factors (IRF; e.g., IRF3 and IRF7). The biological role of TRAFs is largely due to their ability to positively or negatively regulate canonical and non-canonical NF-κB signaling. While TRAF-mediated signaling regulates various immune cell functions, this review is focused on the recent advances in our knowledge regarding the molecular mechanisms through which TRAF proteins regulate, positively and negatively, inflammatory signaling pathways, including Toll–IL-1 receptors, RIG-I like receptors, and Nod-like receptors. The review also offers a perspective on the unanswered questions that need to be addressed to fully understand how TRAFs regulate inflammation.
The poxviruses are large, linear, double-stranded DNA viruses about 130 to 230 kbp, that have an animal origin and evolved to infect a wide host range. Variola virus (VARV), the causative agent of smallpox, is a poxvirus that infects only humans, but other poxviruses such as monkey poxvirus and cowpox virus (CPXV) have crossed over from animals to infect humans. Therefore understanding the biology of poxviruses can devise antiviral strategies to prevent these human infections. In this study we used a system-based approach to examine the host responses to three orthopoxviruses, CPXV, vaccinia virus (VACV), and ectromelia virus (ECTV) in the murine macrophage RAW 264.7 cell line. Overall, we observed a significant down-regulation of gene expressions for pro-inflammatory cytokines, chemokines, and related receptors. There were also common and virus-specific changes in the immune-regulated gene expressions for each poxvirus-infected RAW cells. Collectively our results showed that the murine macrophage RAW 264.7 cell line is a suitable cell-based model system to study poxvirus host response.
Invariant Natural Killer T (iNKT) cells play critical roles in autoimmune, anti‐tumor and anti‐microbial immune responses, and are activated by glycolipids presented by the MHC class I‐like molecule, CD1d. How the activation of signaling pathways impacts antigen (Ag)‐dependent iNKT cell activation is not well‐known. In the current study, we found that the MAPK JNK2 not only negatively regulates CD1d‐mediated Ag presentation in APCs, but also contributes to CD1d‐independent iNKT cell activation. A deficiency in the JNK2 (but not JNK1) isoform enhanced Ag presentation by CD1d. Using a vaccinia virus (VV) infection model known to cause a loss in iNKT cells in a CD1d‐independent, but IL‐12‐dependent manner, we found the virus‐induced loss of iNKT cells in JNK2 KO mice was substantially lower than that observed in JNK1 KO or wildtype (WT) mice. Importantly, compared to WT mice, JNK2 KO mouse iNKT cells were found to express less surface IL‐12 receptors. As with a VV infection, an IL‐12 injection also resulted in a smaller decrease in JNK2 KO iNKT cells as compared to WT mice. Overall, our work strongly suggests JNK2 is a negative regulator of CD1d‐mediated Ag presentation and contributes to IL‐12‐induced iNKT cell activation and loss during viral infections.
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Virus infection is sensed by pattern recognition receptors (PRRs) detecting virus nucleic acids and initiating an innate immune response. DNA-dependent protein kinase (DNA-PK) is a PRR that binds cytosolic DNA and is antagonized by vaccinia virus (VACV) protein C16. Here, VACV protein C4 is also shown to antagonize DNA-PK by binding to Ku and blocking Ku binding to DNA, leading to a reduced production of cytokines and chemokines in vivo and a diminished recruitment of inflammatory cells. C4 and C16 share redundancy in that a double deletion virus has reduced virulence not seen with single deletion viruses following intradermal infection. However, non-redundant functions exist because both single deletion viruses display attenuated virulence compared to wild-type VACV after intranasal infection. It is notable that VACV expresses two proteins to antagonize DNA-PK, but it is not known to target other DNA sensors, emphasizing the importance of this PRR in the response to infection in vivo. DNA-PK is a pattern recognition receptor (PRR) that binds cytosolic DNA and stimulates IRF3 signaling. Scutts et al. show that vaccinia virus antagonizes this DNA sensor with two proteins, C4 and C16, which both block DNA binding.
Host pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs) detect viruses and other pathogens, inducing production of cytokines that cause inflammation and mobilize cells to control infection. Vaccinia virus (VACV) encodes proteins that antagonize these host innate immune responses, and elucidating the mechanisms of action of these viral proteins helped shed light on PRR signalling mechanisms. The VACV virulence factor E3 is one of the most intensely studied VACV proteins and has multiple effects on host cells, many of which cannot be explained by the currently known cellular targets of E3. Here, we report that E3 expression in human monocytes alters TLR2- and TLR8-dependent cytokine induction, and particularly inhibits IL-6. Using MS, we identified DExD/H-box helicase 9 (DHX9) as an E3 target. Although DHX9 has previously been implicated as a PRR for sensing nucleic acid in dendritic cells, we found no role for DHX9 as a nucleic acid-sensing PRR in monocytes. Rather, DHX9 suppression in these cells phenocopied the effects of E3 expression on TLR2- and TLR8-dependent cytokine induction, in that DHX9 was required for all TLR8-dependent cytokines measured, and for TLR2-dependent IL-6. Further, DHX9 also had a cell- and stimulus-independent role in IL-6 promoter induction. DHX9 enhanced nuclear factor kappa B (NFkB)-dependent IL-6 promoter activation, which was directly antagonized by E3. These results indicate new roles for DHX9 in regulating cytokines in innate immunity and reveal that VACV E3 disrupts innate immune responses by targeting of DHX9.
Circulating natural killer (NK) cells help protect the host from lympho-hematogenous acute viral diseases by rapidly entering draining lymph nodes (dLNs) to curb virus dissemination. Here, we identify a highly choreographed mechanism underlying this process. Using footpad infection with ectromelia virus, a pathogenic DNA virus of mice, we show that TLR9/MyD88 sensing induces NKG2D ligands in virus-infected, skin-derived migratory dendritic cells (mDCs) to induce production of IFN-γ by classical NK cells and other types of group 1 innate lymphoid cells (ILCs) already in dLNs, via NKG2D. Uninfected inflammatory monocytes, also recruited to dLNs by mDCs in a TLR9/MyD88-dependent manner, respond to IFN-γ by secreting CXCL9 for optimal CXCR3-dependent recruitment of circulating NK cells. This work unveils a TLR9/MyD88-dependent mechanism whereby in dLNs, three cell types—mDCs, group 1 ILCs (mostly NK cells), and inflammatory monocytes—coordinate the recruitment of protective circulating NK cells to dLNs.
Activation of the DNA-dependent innate immune pathway plays a pivotal role in the host defense against poxvirus. Cyclic GMP-AMP synthase (cGAS) is a key cytosolic DNA sensor that produces the cyclic dinucleotide cGMP-AMP (cGAMP) upon activation, which triggers stimulator of interferon genes (STING), leading to type I Interferons (IFNs) production and an antiviral response. Ectromelia virus (ECTV) has emerged as a valuable model for investigating the host–Orthopoxvirus relationship. However, the role of cGas–Sting pathway in response to ECTV is not clearly understood. Here, we showed that murine cells (L929 and RAW264.7) mount type I IFN responses to ECTV that are dependent upon cGas, Sting, TANK binding kinase 1 (Tbk1), and interferon regulatory factor 3 (Irf3) signaling. Disruption of cGas or Sting expression in mouse macrophages blocked the type I IFN production and facilitated ECTV replication. Consistently, mice deficient in cGas or Sting exhibited lower type I IFN levels and higher viral loads, and are more susceptible to mousepox. Collectively, our study indicates that the cGas–Sting pathway is critical for sensing of ECTV infection, inducing the type I IFN production, and controlling ECTV replication.
Respiratory infection with vaccinia virus (VacV) elicits robust CD8 ⁺ T cell responses that play an important role in host resistance. In the lung, VacV encounters multiple tissue resident APC populations, but which cell plays a dominant role in priming of virus-specific CD8 ⁺ effector T cell responses remains poorly defined. We used Batf3 -/- mice to investigate the impact of CD103 ⁺ DC and CD8α ⁺ DC deficiency on anti-VacV CD8 ⁺ T cell responses. We found that Batf3 -/- mice were more susceptible to VacV infection, exhibiting profound weight loss, which correlated with impaired accumulation of IFN-γ-producing CD8 ⁺ T cell in the lungs. This was largely due to defective priming since early in the response, antigen-specific CD8 ⁺ T cells in the draining lymph nodes of Batf3 -/- mice expressed significantly reduced levels of Ki67, CD25 and T-bet. These results underscore a specific role for Batf3 dependent DCs in regulating priming and expansion of effector CD8 ⁺ T cells necessary for host resistance against acute respiratory VacV infection.
IMPORTANCE During respiratory infection with vaccinia virus (VacV), a member of Poxviridae family, CD8 ⁺ T cells play important role in resolving the primary infection. Effector CD8 ⁺ T cells clear the virus by accumulating in the infected lungs in high numbers and secreting molecules such as IFN-γ that kill virally infected cells. However, precise cell-types that regulate the generation of effector CD8 ⁺ T cells in the lungs are not well defined. Dendritic cells (DCs) are heterogeneous population of immune cells that are recognized as key initiators and regulators of T cell mediated immunity. In this study, we reveal that a specific subset of DCs that are dependent on the transcription factor, Batf3, for their development, regulate the magnitude of CD8 ⁺ T cell effector responses in the lungs, thereby providing protection during pulmonary VacV infection.
The largest outbreak of monkeypox in history began in May, 2022, and has rapidly spread across the globe ever since. The purpose of this Review is to briefly describe human immune responses to orthopoxviruses; provide an overview of the vaccines available to combat this outbreak; and discuss the various clinical data and animal studies evaluating protective immunity to monkeypox elicited by vaccinia virus-based smallpox vaccines, address ongoing concerns regarding the outbreak, and provide suggestions for the appropriate use of vaccines as an outbreak control measure. Data showing clinical effectiveness (~85%) of smallpox vaccines against monkeypox come from surveillance studies conducted in central Africa in the 1980s and later during outbreaks in the same area. These data are supported by a large number of animal studies (primarily in non-human primates) with live virus challenge by various inoculation routes. These studies uniformly showed a high degree of protection and immunity against monkeypox virus following vaccination with various smallpox vaccines. Smallpox vaccines represent an effective countermeasure that can be used to control monkeypox outbreaks. However, smallpox vaccines do cause side-effects and the replication-competent, second-generation vaccines have contraindications. Third-generation vaccines, although safer for use in immunocompromised populations, require two doses, which is an impediment to rapid outbreak response. Lessons learned from the COVID-19 pandemic should be used to inform our collective response to this monkeypox outbreak and to future outbreaks.
Orf virus (ORFV) is the type species of the Parapoxvirus genus that belongs to the Poxviridae family. Type I interferons (IFN) are critical in the host defence against viruses. They induce hundreds of interferon stimulated genes (ISGs) many of which have an antiviral role. The ability of ORFV to modulate type I IFN production was undertaken to investigate whether ORFV could inhibit IFN-β expression via dsRNA dependant signalling pathways. HEK293 cells are known to lack DNA pattern-recognition receptors and Toll-like receptors however, they do express the cytosolic dsRNA receptors RIG-I and MDA5. HEK293 cells were shown to produce high levels of IFN-β when cells were stimulated with poly(I:C) and this was shown to be predominantly via RIG-I-dependent signalling as confirmed by siRNA knock-down of RIG-I. Further we showed that HEK293 cells are permissive for ORFV and caused potent inhibition of IFN-β transcription when cells were stimulated with poly(I:C) post-viral infection. Studies using heat inactivated ORFV suggested that de novo synthesis of early genes was required. In addition our findings showed that the ORFV encoded factor ORF020, that is known to bind dsRNA, is involved in antagonising IFN expression. Overall, this study has shown for first time the ability of ORFV to counteract type I IFN expression by antagonising dsRNA-activated RIG-I signalling.
Z-DNA Binding protein 1 (ZBP1) activates Receptor Interacting Protein Kinase 3 (RIPK3) -dependent cell death during lytic infection by members of the orthomyxovirus, herpesvirus and poxvirus families. ZBP1 possesses two Zα domains capable of selective binding to Z-DNA, as well as to Z-RNA. We have now unveiled Z-RNA as the ligand that activates ZBP1 in cells infected with orthomyxoviruses (influenza A and B viruses) and the poxvirus vaccinia virus (VACV). Orthomyxovirus Z-RNA is sensed by ZBP1 in the nucleus of infected cells, resulting in nuclear activation of RIPK3, consequent rupture of the nucleus, and hyper-inflammatory ‘nuclear necroptosis’. VACV-generated Z-RNA accumulates in the cytoplasm, where it is sequestered from ZBP1 by E3, the viral E3L gene product. In viruses where the E3 Zα domain has been mutated, ZBP1 senses Z-RNA and triggers RIPK3-dependent necroptosis in the cytoplasm. Z-RNA is thus a new viral pathogen-associated molecular pattern (PAMP).
Necroptosis mediated by Z-nucleic-acid-binding protein (ZBP)1 (also called DAI or DLM1) contributes to innate host defense against viruses by triggering cell death to eliminate infected cells. During infection, vaccinia virus (VACV) protein E3 prevents death signaling by competing for Z-form RNA through an N-terminal Zα domain. In the absence of this E3 domain, Z-form RNA accumulates during the early phase of VACV infection, triggering ZBP1 to recruit receptor interacting protein kinase (RIPK)3 and execute necroptosis. The C-terminal E3 double-strand RNA-binding domain must be retained to observe accumulation of Z-form RNA and induction of necroptosis. Substitutions of Zα from either ZBP1 or the RNA-editing enzyme double-stranded RNA adenosine deaminase (ADAR)1 yields fully functional E3 capable of suppressing virus-induced necroptosis. Overall, our evidence reveals the importance of Z-form RNA generated during VACV infection as a pathogen-associated molecular pattern (PAMP) unleashing ZBP1/RIPK3/MLKL-dependent necroptosis unless suppressed by viral E3.
Natural killer (NK) cells are innate lymphocytes that provide critical host defense against pathogens and cancer. Originally heralded for their early and rapid effector activity, NK cells have been recognized over the last decade for their ability to undergo adaptive immune processes, including antigen-driven clonal expansion and generation of long-lived memory. This review presents an overview of how NK cells lithely partake in both innate and adaptive responses and how this versatility is manifest in human NK cell-mediated immunity.
Loss of appetite (anorexia) is a typical behavioral response to infectious diseases that often reduces body weight. Also, anorexia can be observed in cancer and trauma patients, causing poor quality of life and reduced prospects of positive therapeutic outcomes. Although anorexia is an acute symptom, its initiation and endocrine regulation during antiviral immune responses are poorly understood. During viral infections, plasmacytoid dendritic cells (pDCs) produce abundant type I interferon (IFN-I) to initiate first-line defense mechanisms. Here, by targeted ablation of pDCs and various in vitro and in vivo mouse models of viral infection and inflammation, we identified that IFN-I is a significant driver of somatostatin (SST). Consequently, SST suppressed the hunger hormone ghrelin that led to severe metabolic changes, anorexia, and rapid bodyweight loss. Furthermore, during vaccination with Modified Vaccinia Ankara virus (MVA), the SST-mediated suppression of ghrelin was critical to viral immune response, as ghrelin restrained the production of early cytokines by natural killer (NK) cells and pDCs, and impaired the clonal expansion of CD8+ T cells. Thus, the hormonal modulation of ghrelin through SST and the cytokine IFN-I is fundamental for optimal antiviral immunity, which comes at the expense of calorie intake.
Activation of the cyclic guanosine monophosphate (GMP)-AMP (cGAMP) sensor STING requires its translocation from the endoplasmic reticulum to the Golgi apparatus and subsequent polymerization. Using a genome-wide CRISPR-Cas9 screen to define factors critical for STING activation in cells, we identified proteins critical for biosynthesis of sulfated glycosaminoglycans (sGAGs) in the Golgi apparatus. Binding of sGAGs promoted STING polymerization through luminal, positively charged, polar residues. These residues are evolutionarily conserved, and selective mutation of specific residues inhibited STING activation. Purified or chemically synthesized sGAGs induced STING polymerization and activation of the kinase TBK1. The chain length and O-linked sulfation of sGAGs directly affected the level of STING polymerization and, therefore, its activation. Reducing the expression of Slc35b2 to inhibit GAG sulfation in mice impaired responses to vaccinia virus infection. Thus, sGAGs in the Golgi apparatus are necessary and sufficient to drive STING polymerization, providing a mechanistic understanding of the requirement for endoplasmic reticulum (ER)-to-Golgi apparatus translocation for STING activation.
Many poxviruses are pathogens affecting humans or animals. Smallpox was one of the worst infectious diseases in human history and remains a bioterrorism concern despite its eradication from nature. Animal poxviruses cause significant economic losses for agricultural industries and are a source for zoonotic outbreaks. Poxviruses have complex virions and are considerably self-sufficient, thanks to a large genome coding for many proteins that regulate both viral and cellular processes. The study of this family of viruses has contributed greatly to our understanding of viral replication and host-pathogen interaction. Here we give a broad overview of the Poxviridae family, including details on its structure, genome, replication cycle, immune response, and immune evasion mechanisms.
Retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) are key sensors of virus infection, mediating the transcriptional induction of type I interferons and other genes that collectively establish an antiviral host response. Recent studies have revealed that both viral and host-derived RNAs can trigger RLR activation; this can lead to an effective antiviral response but also immunopathology if RLR activities are uncontrolled. In this Review, we discuss recent advances in our understanding of the types of RNA sensed by RLRs in the contexts of viral infection, malignancies and autoimmune diseases. We further describe how the activity of RLRs is controlled by host regulatory mechanisms, including RLR-interacting proteins, post-translational modifications and non-coding RNAs. Finally, we discuss key outstanding questions in the RLR field, including how our knowledge of RLR biology could be translated into new therapeutics.
Viral infection triggers host defenses through pattern-recognition receptor-mediated cytokine production, inflammasome activation, and apoptosis of the infected cells. Inflammasome-activated caspases are known to cleave cyclic GMP-AMP synthase (cGAS). Here, we found that apoptotic caspases are critically involved in regulating both DNA and RNA virus-triggered host defenses, in which activated caspase-3 cleaved cGAS, MAVS, and IRF3 to prevent cytokine overproduction. Caspase-3 was exclusively required in human cells, whereas caspase-7 was involved only in murine cells to inactivate cGAS, reflecting distinct regulatory mechanisms in different species. Caspase-mediated cGAS cleavage was enhanced in the presence of dsDNA. Alternative MAVS cleavage sites were used to ensure the inactivation of this critical protein. Elevated type I IFNs were detected in caspase-3-deficient cells without any infection. Casp3 −/− mice consistently showed increased resistance to viral infection and experimental autoimmune encephalomyelitis. Our results demonstrate that apoptotic caspases control innate immunity and maintain immune homeostasis against viral infection. Ning et al. find that caspase-3 cleaves and inactivates cGAS, MAVS, and IRF3 to suppress cytokine and type I IFN production. Their findings reveal a role for apoptotic caspases in controlling antiviral innate immunity and keeping apoptosis immunologically silent.
The mononuclear phagocyte system (MPS) is defined as a cell lineage in which committed marrow progenitors give rise to blood monocytes and tissue macrophages. Here, we discuss the concept of self-proscribed macrophage territories and homeostatic regulation of tissue macrophage abundance through growth factor availability. Recent studies have questioned the validity of the MPS model and argued that tissue-resident macrophages are a separate lineage seeded during development and maintained by self-renewal. We address this issue; discuss the limitations of inbred mouse models of monocyte-macrophage homeostasis; and summarize the evidence suggesting that during postnatal life, monocytes can replace resident macrophages in all major organs and adopt their tissue-specific gene expression. We conclude that the MPS remains a valid and accurate framework for understanding macrophage development and homeostasis.
Viruses employ elaborate strategies to coopt the cellular processes they require to replicate while simultaneously thwarting host antiviral responses. In many instances, how this is accomplished remains poorly understood. Here, we identify a protein, F17 encoded by cytoplasmically replicating poxviruses, that binds and sequesters Raptor and Rictor, regulators of mammalian target of rapamycin complexes mTORC1 and mTORC2, respectively. This disrupts mTORC1-mTORC2 crosstalk that coordinates host responses to poxvirus infection. During infection with poxvirus lacking F17, cGAS accumulates together with endoplasmic reticulum vesicles around the Golgi, where activated STING puncta form, leading to interferon-stimulated gene expression. By contrast, poxvirus expressing F17 dysregulates mTOR, which localizes to the Golgi and blocks these antiviral responses in part through mTOR-dependent cGAS degradation. Ancestral conservation of Raptor/Rictor across eukaryotes, along with expression of F17 across poxviruses, suggests that mTOR dysregulation forms a conserved poxvirus strategy to counter cytosolic sensing while maintaining the metabolic benefits of mTOR activity.
Cytosolic recognition of DNA has emerged as a critical cellular mechanism of host immune activation upon pathogen invasion. The central cytosolic DNA sensor cGAS activates STING, which is phosphorylated, dimerizes and translocates from the endoplasmic reticulum (ER) to a perinuclear region to mediate IRF-3 activation. Poxviruses are double-stranded DNA viruses replicating in the cytosol and hence likely to trigger cytosolic DNA sensing. Here, we investigated the activation of innate immune signaling by 4 different strains of the prototypic poxvirus vaccinia virus (VACV) in a cell line proficient in DNA sensing. Infection with the attenuated VACV strain MVA activated IRF-3 via cGAS and STING, and accordingly STING dimerized and was phosphorylated during MVA infection. Conversely, VACV strains Copenhagen and Western Reserve inhibited STING dimerization and phosphorylation during infection and in response to transfected DNA and cyclic GMP-AMP, thus efficiently suppressing DNA sensing and IRF-3 activation. A VACV deletion mutant lacking protein C16, thought to be the only viral DNA sensing inhibitor acting upstream of STING, retained the ability to block STING activation. Similar inhibition of DNAinduced STING activation was also observed for cowpox and ectromelia viruses. Our data demonstrate that virulent poxviruses possess mechanisms for targeting DNA sensing at the level of the cGAS-STING axis and that these mechanisms do not operate in replication-defective strains such as MVA. These findings shed light on the role of cellular DNA sensing in poxvirus-host interactions and will open new avenues to determine its impact on VACV immunogenicity and virulence.