Vaccinia Virus Infection Research Articles

Vaccinia virus infection suppresses the cell microRNA machinery

MicroRNAs are key players in the regulation of gene expression by posttranscriptional suppression. They are involved in physiological processes, and thus their deregulation may contribute to the development of diseases and progression of cancer. Virus-encoded microRNAs and microRNAs of host origin play an important role in controlling the virus life cycle and immunity. The aim of this study was to determine the effect of vaccinia virus (VACV) infection on the expression of host-encoded microRNAs. A marked general suppression of most microRNAs in the infected cells was observed within 24 hours after VACV infection of a number of cell types. We demonstrate that this suppression was associated with abrogation of expression of the Dicer1 enzyme, which is a key enzyme in the generation of microRNAs.

Poxvirus Infection-Associated Downregulation of C-Type Lectin-Related-b Prevents NK Cell Inhibition by NK Receptor Protein-1B

Innate immune recognition of virus-infected cells includes NK cell detection of changes to endogenous cell-surface proteins through inhibitory receptors. One such receptor system is the NK cell receptor protein-1B (NKR-P1B) and its ligand C-type lectin-related-b (Clr-b). NKR-P1B and Clr-b are encoded within the NK cell gene complex, a locus that has been linked to strain-dependent differences in susceptibility to infection by poxviruses. In this study, we report the impact of vaccinia virus (VV) and ectromelia virus infection on expression of Clr-b and Clr-b-mediated protection from NK cells. We observed a loss of Clr-b cell-surface protein upon VV and ectromelia virus infection of murine cell lines and bone marrow-derived macrophages. The reduction of Clr-b is more rapid than MHC class I, the prototypic ligand of NK cell inhibitory receptors. Reduction of Clr-b requires active viral infection but not expression of late viral genes, and loss of mRNA appears to lag behind loss of Clr-b surface protein. Clr-b-mediated protection from NK cells is lost following VV infection. Together, these results provide the second example of Clr-b modulation during viral infection and suggest reductions of Clr-b may be involved in sensitizing poxvirus-infected cells to NK cells.

Allergic lung inflammation decreases the anti-viral response to vaccinia virus (175.3)

Abstract Despite the global eradication of smallpox following widespread vaccination with vaccinia virus (VV), other members of the poxviridae family persist and continue to infect animal and human hosts. Although the lung is the most lethal route of poxvirus infection, there is little known about the local immune response to pulmonary VV infection. Moreover, VV vaccination is contraindicated in patients with atopic dermatitis as the pro-allergic milieu diminishes anti-VV responses. Our goal was to determine the role of allergic airway disease (AAD) on the local immune response to VV infection. Our studies demonstrated increased weight loss, airway hyperreactivity, mucus gene expression, lung pathology and virus titer in mice with AAD. In contrast to observations from cells exposed to pro-allergic cytokines, expression of antimicrobial peptide genes in the lung tissue of infected mice was similar in control mice, and mice with AAD. At 4 days post infection (dpi) there was a more robust innate response in mice with AAD as evidenced by increased infiltration of lung macrophages and granulocytes. By 10 dpi, there were increased numbers of IL-10+CD8+ and IFNγ+CD8+ T cells and enhanced secretion of IL-10 and IFNγ in the bronchoalveolar lavage fluid of mice with AAD, compared to controls. Mice with AAD also had increased VV-specific IgG1 and IgM serum antibodies. These studies suggest the allergic lung microenvironment may influence the anti-viral immune response to pulmonary VV infection.

Retinoic acid modulation of CD8 effector and memory differentiation. (110.4)

Abstract A plethora of work implicates important effects of the Vitamin A derivative, retinoic acid (RA), in myeloid differentiation, while fewer studies explore the role of RA on lymphoid cells. Most work has looked at the role of RA on CD4 T cells, focusing on regulatory CD4 T cell differentiation and maintenance of this phenotype along with some Th1 functions. There is little information about the role of RA in CD8 T cell differentiation, and even less on cell-intrinsic effects in the CD8 T cell. In this study we explored the role of RA on effector and memory differentiation in a direct and cell intrinsic manner in the context of vaccinia virus infection. We used dominant negative RA alpha-receptor (RARα DN) expressing mice and bone marrow chimeric mice containing both WT and RARα DN compartments. We observed the loss of the short-lived effector cell phenotype and an enhanced memory precursor phenotype at the effector and contraction phase of the CD8 response in the absence of RA signaling. Further, the lack of RA increased the proportion of central memory CD8s. Vitamin A is used in conjunction with various vaccines (e.g. Measles vaccine), these results will elucidate the effect of RA in shaping the effector and memory response helping to tailor the supplement to better enhance the outcome of vaccination.

Interruption with TNFα/TNFR1 function impairs host immune response against cutaneous vaccinia virus infection (52.8)

Abstract One strategy adopted by vaccinia virus (VV) to evade the host immune system is to encode homologs of TNF receptors (TNFR) that block TNFα function. The response to VV skin infection under conditions of TNFα deficiency, however, has not been reported. We found that TNFR1-/- developed larger skin lesions with higher virus levels after VV scarification. Following their recovery, these TNFR1-/- mice were fully protected against lethal VV intranasal challenge, suggesting their effective memory immune response. A functional systemic immune response of TNFR1-/- mice was further demonstrated by enhanced production of VV-specific IFNγ and CD8+ T cells in spleens and draining lymph nodes as well as efficient T cell skin homing. Interestingly, bone marrow (BM) reconstitution studies using WT BM →TNFR1-/- mice, but not TNFR1-/- BM → WT mice, reproduced the original results seen in TNFR1-/- mice, indicating that TNFR1 deficiency in resident skin cells, rather than hematopoietic cells, accounts for the impaired cutaneous immune response. Similar phenotype was also found in in TNFα-/- mice following VV scarification while both WT BM→ TNFα-/- mice and TNFα-/-BM →WT mice developed significantly larger skin lesions with higher virus counts than the control chimeric mice.Therefore, while the target of TNFα action is restricted to skin stromal components, the source of TNFα produced in response to VV infection would appear to include both hematopoietic cells and radioresistant skin elements.

Disruption of TNF-α/TNFR1 Function in Resident Skin Cells Impairs Host Immune Response against Cutaneous Vaccinia Virus Infection

One strategy adopted by vaccinia virus (VV) to evade the host immune system is to encode homologs of TNF receptors (TNFR) that block TNFα function. The response to VV skin infection under conditions of TNFα deficiency, however, has not been reported. We found that TNFR1−/− mice developed larger primary lesions, numerous satellite lesions and higher skin virus levels after VV scarification. Following their recovery, these TNFR1−/− mice were fully protected against challenge with a lethal intranasal dose of VV, suggesting these mice developed an effective memory immune response. A functional systemic immune response of TNFR1−/− mice was further demonstrated by enhanced production of VV-specific IFNγ and VV-specific CD8+ T cells in spleens and draining lymph nodes. Interestingly, bone marrow (BM) reconstitution studies using WT BM in TNFR1−/− host mice, but not TNFR1−/− BM in WT host mice, reproduced the original results seen in TNFR1−/− mice, indicating that TNFR1 deficiency in resident skin cells, rather than hematopoietic cells, accounts for the impaired cutaneous immune response. Our data suggest that lack of TNFR1 leads to a skin-specific immune deficiency and that resident skin cells play a crucial role in mediating an optimal immune defense to VV cutaneous infection via TNFα/TNFR1 signaling.

Position on donors and smallpox vaccine: a committee opinion

Although there is presently no definitive evidence linking vaccinia virus transmission through reproductive cells, SART/ASRM accordingly recommends that ART practitioners consider deferring donors who have recently received smallpox vaccine or contracted symptomatic vaccinia virus infection through close contact with a vaccine recipient (until after the vaccine or infectious scab has spontaneously separated). Good donor practice further suggests that donors who are not in good health, including those with recent complications from smallpox vaccine, should be similarly deferred. (This document was reviewed by the ASRM Practice Committee in 2011).

Loss of Cytoskeletal Transport during Egress Critically Attenuates Ectromelia Virus Infection In Vivo

Egress of wrapped virus (WV) to the cell periphery following vaccinia virus (VACV) replication is dependent on interactions with the microtubule motor complex kinesin-1 and is mediated by the viral envelope protein A36. Here we report that ectromelia virus (ECTV), a related orthopoxvirus and the causative agent of mousepox, encodes an A36 homologue (ECTV-Mos-142) that is highly conserved despite a large truncation at the C terminus. Deleting the ECTV A36R gene leads to a reduction in the number of extracellular viruses formed and to a reduced plaque size, consistent with a role in microtubule transport. We also observed a complete loss of virus-associated actin comets, another phenotype dependent on A36 expression during VACV infection. ECTV ΔA36R was severely attenuated when used to infect the normally susceptible BALB/c mouse strain. ECTV ΔA36R replication and spread from the draining lymph nodes to the liver and spleen were significantly reduced in BALB/c mice and in Rag-1-deficient mice, which lack T and B lymphocytes. The dramatic reduction in ECTV ΔA36R titers early during the course of infection was not associated with an augmented immune response. Taken together, these findings demonstrate the critical role that subcellular transport pathways play not only in orthopoxvirus infection in an in vitro context but also during orthopoxvirus pathogenesis in a natural host. Furthermore, despite the attenuation of the mutant virus, we found that infection nonetheless induced protective immunity in mice, suggesting that orthopoxvirus vectors with A36 deletions may be considered another safe vaccine alternative.

The DC receptor DNGR-1 mediates cross-priming of CTLs during vaccinia virus infection in mice

In order to prime T cells, DCs integrate signals emanating directly from pathogens and from their noxious action on the host. DNGR-1 (CLEC9A) is a DC-restricted receptor that detects dead cells. Therefore, we investigated the possibility that DNGR-1 affects immunity to cytopathic viruses. DNGR-1 was essential for cross-presentation of dying vaccinia virus-infected (VACV-infected) cells to CD8(+) T cells in vitro. Following injection of VACV or VACV-infected cells into mice, DNGR-1 detected the ligand in dying infected cells and mediated cross-priming of anti-VACV CD8(+) T cells. Loss of DNGR-1 impaired the CD8+ cytotoxic response to VACV, especially against those virus strains that are most dependent on cross-presentation. The decrease in total anti-VACV CTL activity was associated with a profound increase in viral load and delayed resolution of the primary lesion. In addition, lack of DNGR-1 markedly diminished protection from infection induced by vaccination with the modified vaccinia Ankara (MVA) strain. DNGR-1 thus contributes to anti-VACV immunity, following both primary infection and vaccination. The non-redundant ability of DNGR-1 to regulate cross-presentation of viral antigens suggests that this form of regulation of antiviral immunity could be exploited for vaccination.

Increased Protection from Vaccinia Virus Infection in Mice Genetically Prone to Lymphoproliferative Disorders

Mutations in the genes that encode Fas or Fas ligand (FasL) can result in poor restraints on lymphocyte activation and in increased susceptibility to autoimmune disorders. Because these mutations portend a continuously activated immune state, we hypothesized that they might in some cases confer resistance to infection. To examine this possibility, the immune response to, morbidity caused by, and clearance of vaccinia virus (VACV) Western Reserve was examined in 5- to 7-week-old Fas mutant (lpr) mice, before an overt lymphoproliferative disorder was observable. On day 6 after VACV infection, C57BL/6-lpr (B6-lpr) mice had decreased morbidity, decreased viral titers, and an increased percentage and number of CD4(+) and CD8(+) T cells. As early as day 2 after infection, B6-lpr mice had decreased liver and spleen viral titers and increased numbers of and increased gamma interferon (IFN-γ) production by several different effector cell populations. Depletion of individual effector cell subsets did not inhibit the resistance of B6-lpr mice. Uninfected B6-lpr mice also had increased numbers of NK cells, γδ(+) T cells, and CD44(+) CD4(+) and CD44(+) CD8(+) T cells compared to uninfected B6 mice. Antibody to IFN-γ resulted in increased virus load in both B6 and B6-lpr mice and eliminated the differences in viral titers between them. These results suggest that IFN-γ produced by multiple activated leukocyte populations in Fas-deficient hosts enhances resistance to some viral infections.

Skin infection generates non-migratory memory CD8+ TRM cells providing global skin immunity

Protective T-cell memory has long been thought to reside in blood and lymph nodes, but recently the concept of immune memory in peripheral tissues mediated by resident memory T (T(RM)) cells has been proposed. Here we show in mice that localized vaccinia virus (VACV) skin infection generates long-lived non-recirculating CD8(+) skin T(RM) cells that reside within the entire skin. These skin T(RM) cells are potent effector cells, and are superior to circulating central memory T (T(CM)) cells at providing rapid long-term protection against cutaneous re-infection. We find that CD8(+) T cells are rapidly recruited to skin after acute VACV infection. CD8(+) T-cell recruitment to skin is independent of CD4(+) T cells and interferon-γ, but requires the expression of E- and P-selectin ligands by CD8(+) T cells. Using parabiotic mice, we further show that circulating CD8(+) T(CM) and CD8(+) skin T(RM) cells are both generated after skin infection; however, CD8(+) T(CM) cells recirculate between blood and lymph nodes whereas T(RM) cells remain in the skin. Cutaneous CD8(+) T(RM) cells produce effector cytokines and persist for at least 6 months after infection. Mice with CD8(+) skin T(RM) cells rapidly cleared a subsequent re-infection with VACV whereas mice with circulating T(CM) but no skin T(RM) cells showed greatly impaired viral clearance, indicating that T(RM) cells provide superior protection. Finally, we show that T(RM) cells generated as a result of localized VACV skin infection reside not only in the site of infection, but also populate the entire skin surface and remain present for many months. Repeated re-infections lead to progressive accumulation of highly protective T(RM) cells in non-involved skin. These findings have important implications for our understanding of protective immune memory at epithelial interfaces with the environment, and suggest novel strategies for vaccines that protect against tissue tropic organisms.

Validation of an immunoperoxidase monolayer assay for total anti–Vaccinia virus antibody titration

Vaccinia virus (VACV) has been associated with zoonotic exanthemic outbreaks affecting bovids and human beings, with significant public health and economic impacts. Rapid and reliable diagnostic methods are needed to detect and epidemiologically monitor antibodies to VACV. The current study describes the development of an immunoperoxidase monolayer assay (IPMA) for detection of total VACV antibodies in bovine serum. The assay was validated by comparison with a plaque reduction neutralization test (PRNT). Kappa index of agreement, diagnostic sensitivity, specificity, and accuracy of the IPMA were -1.008, 100%, 96%, and 98%, respectively, when compared with PRNT on 148 field bovine sera. Repeatability tests on 32 field-positive serum samples revealed that intraclass coefficient correlation was 0.86. In experimentally infected cattle, VACV antibodies were detectable by IPMA 4 days postinfection, which was more than 2 weeks earlier than with the PRNT, indicating that IPMA could be a more sensitive test than the latter. In 4 naturally VACV-diseased cows monitored for 13 months, IPMA could detect VACV antibodies up to 13 months, a longer time than PRNT. The IPMA is simpler to produce and perform when compared with PRNT and is time saving and suitable for large-scale surveys of VACV infection in bovine.

Cutting Edge: Protective Effect of CX3CR1+ Dendritic Cells in a Vaccinia Virus Pulmonary Infection Model

The protective host immune response to viral infections requires both effective innate and adaptive immune responses. Cross-talk between the two responses is coordinated by the chemokine network and professional APCs such as dendritic cells (DCs). In mice, subpopulations of myeloid DCs in peripheral tissues such as lungs and in blood express CX3CR1 depending on the inflammation state. We thus examined the host response of mice deficient in the chemokine receptor CX3CR1 to an intranasal vaccinia virus infection. CX3CR1-deficient mice displayed significantly more severe morbidity and mortality compared with control wild-type mice within 10 d following vaccinia virus infection. CX3CR1(-/-) mice had increased viral loads and a reduced T cell response compared with wild-type mice. Finally, an adoptive transfer of CX3CR1(+/+) DCs completely protected CX3CR1(-/-) mice to a previously lethal infection. This study therefore opens up the possibility of novel antiviral therapeutics targeting lung DC recruitment.

Staphylococcus Aureus Alpha-Toxin Augments Viral Load In Keratinocytes

The factor(s) leading to increase propensity of disseminated herpes simplex virus (HSV) and vaccinia virus (VV) infections in atopic dermatitis (AD) are poorly understood. A history of eczema herpeticum is frequently associated with Staphylococcus aureus infection. This study investigated whether S. aureus toxins augment viral load in normal human keratinocytes (NHK).

C3a Receptor (C3aR) is Important for Viral Containment in Mice Inoculated with Vaccinia Virus (VV) at Sites of Allergic Skin Inflammation

Eczema Vaccinatum (EV) is a life-threatening complication of exposure to smallpox vaccination in patients with atopic dermatitis (AD). We previously showed that C3aR functions to downregulate local and systemic Th2 immune response to epicutaneously (EC) introduced antigen, and that a cutaneous Th2 response at the VV inoculation site impairs virus containment. We examined whether C3aR confers resistance to cutaneous VV infection.

Glomulin: A Permissivity Factor for Vaccinia Virus Infection

In earlier studies we provided evidence that vaccinia virus (VACV) phosphorylation-activation of host cell signaling effectors is critical for subsequent viral replication. In this report, using mass spectrometry-based proteomics, we have identified 387 host cell proteins that co-immunoprecipitate with VACV in infected, permissive PM1.CCR5 human T cells. Among these, glomulin was distinguishable based on its known interaction with a tyrosine kinase receptor, c-Met, its ability to become tyrosine-phosphorylated, and its association with signaling effectors. siRNA knockdown of glomulin expression in PM1.CCR5 T cells reduces VACV infection. Glomulin interacts with the inactive, nonphosphorylated form of c-MET. We demonstrate that treatment of PM1.CCR5 T cells with a c-Met phosphorylation inhibitor leads to a significant reduction in VACV infectivity. Additionally, inhibition of phosphorylation of c-Met abrogates VACV-inducible phosphorylation of Erk 1/2 and IRS-2, signaling effectors identified as critical for VACV infection. These data identify glomulin as a permissivity factor for VACV infection and as a potential therapeutic target for inhibition of VACV infection.

Susceptibility to Vaccinia Virus Infection and Spread in Mice Is Determined by Age at Infection, Allergen Sensitization and Mast Cell Status

Background: Patients, especially young children, with atopic dermatitis are at an increased risk of developing eczema vaccinatum, a severe reaction to the smallpox vaccine, either through direct vaccination or indirect contact with a person recently vaccinated. Methods: Using a mouse model of infection, the severity of vaccinia-induced lesions was assessed from their appearance and viral DNA content. The response to vaccinia inoculation was assessed in young and adult mice, allergen-sensitized mice, and in mast cell-deficient mice. Results: Young age, sensitization to an allergen prior to infection, and a mast cell deficit, accomplished by using mast cell-deficient mice, resulted in more severe viral lesions at the site of inoculation, according to lesion appearance and viral DNA content. All three factors combined demonstrated maximal susceptibility, characterized by the severity of primary lesions and the development of secondary (satellite) lesions, as occurs in eczema vaccinatum in humans. Resistance to the appearance of satellite lesions could be restored by adoptive transfer of bone marrow-derived mast cells from either wild-type or cathelicidin-related antimicrobial peptide-deficient mice. Primary lesions were more severe following the latter transfer, indicating that cathelicidin-related antimicrobial peptide does contribute to the protective activity of mast cells against infection. Conclusions: The combination of young age, allergen sensitization and a mast cell deficit resulted in the most severe lesions, including satellite lesions. Understanding the factors determining the relative resistance/sensitivity to vaccinia virus will aid in the development of strategies for preventing and treating adverse reactions which can occur after smallpox vaccination.

Vaccinia viruses isolated from skin infection in horses produced cutaneous and systemic disease in experimentally infected rabbits

The susceptibility of rabbits to two isolates of Vaccinia virus (VACV) recovered from cutaneous disease in horses in Southern Brazil was investigated. Rabbits were inoculated in the ear skin with both VACV isolates, either in single or mixed infection. All inoculated animals presented local skin lesions characterized by hyperaemia, papules, vesicles, pustules and ulcers. Infectious virus was detected in the lungs and intestine of rabbits that died during acute disease. Histological examination of the skin revealed changes characteristic of those associated with members of the genus Orthopoxvirus. These results demonstrate that rabbits develop skin disease accompanied by systemic signs upon intradermal inoculation of these two equine VACV isolates, either alone or in combination, opening the way for using rabbits to study selected aspects of the biology and pathogenesis of VACV infection.

Skin Mast Cells Protect Mice against Vaccinia Virus by Triggering Mast Cell Receptor S1PR2 and Releasing Antimicrobial Peptides

Mast cells (MCs) are well-known effectors of allergic reactions and are considered sentinels in the skin and mucosa. In addition, through their production of cathelicidin, MCs have the capacity to oppose invading pathogens. We therefore hypothesized that MCs could act as sentinels in the skin against viral infections using antimicrobial peptides. In this study, we demonstrate that MCs react to vaccinia virus (VV) and degranulate using a membrane-activated pathway that leads to antimicrobial peptide discharge and virus inactivation. This finding was supported using a mouse model of viral infection. MC-deficient (Kit(wsh-/-)) mice were more susceptible to skin VV infection than the wild type animals, whereas Kit(wsh-/-) mice reconstituted with MCs in the skin showed a normal response to VV. Using MCs derived from mice deficient in cathelicidin antimicrobial peptide, we showed that antimicrobial peptides are one important antiviral granule component in in vivo skin infections. In conclusion, we demonstrate that MC presence protects mice from VV skin infection, MC degranulation is required for protecting mice from VV, neutralizing Ab to the L1 fusion entry protein of VV inhibits degranulation apparently by preventing S1PR2 activation by viral membrane lipids, and antimicrobial peptide release from MC granules is necessary to inactivate VV infectivity.

Casein kinase 2 regulates vaccinia virus actin tail formation

Casein kinase 2 (CK2) is a pleiotropic serine/threonine kinase that regulates numerous cellular processes and is essential to the infectious cycle of several viruses. Here we investigated the potential role of CK2 in vaccinia virus (VACV) infection. We used the CK2 inhibitor TBB and found that CK2 inactivation impaired VACV dissemination and actin tail formation. We used RNAi and confirmed that CK2 depletion impaired VACV actin tail formation. Furthermore, we designed a recombinant virus that allowed us to specifically detect cell-associated enveloped viruses (CEVs) at the plasma membrane and demonstrated that CK2 inactivation does not affect CEV formation. Finally, we showed that CK2 depletion impaired the recruitment of Src to CEVs. We discuss the possibility that CK2 may stimulate the A36-dependent recruitment of Src through A36 phosphorylation.