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Non-typhoidal Salmonellae (NTS) are common foodborne pathogens throughout the world causing acute gastroenteritis. Compared to North America and Europe, there is little information on NTS in the Caribbean. Here we investigated the prevalence and characteristics of NTS present in the local poultry of the Cayman Islands to determine the public health risk. In total, we collected 156 samples. These were made up of boot swabs of 31 broiler farms and 31 layer farms (62 samples), paper bedding from 45 imported chick boxes, and 49 pooled cecum samples from feral chickens, each sample representing 10 individual chickens. Salmonella was isolated using the ISO 6579 protocol and isolates were characterized using Whole Genome Sequencing (WGS) analysis. Eighteen Salmonella isolates were obtained and comprised six S. enterica subspecies enterica serotypes and one subspecies houtenae serotype. Serotypes were: S. Kentucky ( n = 9), S. Saintpaul ( n = 5), S. Javiana ( n = 1), S. Senftenberg ( n = 1), S. Poona ( n = 1) and S. Agona ( n = 1). S . Kentucky strains were all ST152 and clonally related to poultry strains from the United states. S . Saintpaul ST50 strains showed clonality to North American strains. Over half of the strains ( n = 11) contained resistance genes to at least two antibiotic groups and five strains were MDR, mainly those from imported day-old chicks. The bla CMY-2 gene was found in S. Kentucky from day-old chicks. Strains from feral poultry had no acquired AMR genes. While serotypes from feral poultry have been identified in human infections, they pose minimal risk due to their low virulence., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Watler, Toka, Lardé, Johnson and Butaye.)

Ectromelia virus (ECTV) is a causative agent of mousepox. It provides a suitable model for studying the immunobiology of orthopoxviruses, including their interaction with the host cell cytoskeleton. As professional antigen-presenting cells, dendritic cells (DCs) control the pericellular environment, capture antigens, and present them to T lymphocytes after migration to secondary lymphoid organs. Migration of immature DCs is possible due to the presence of specialized adhesion structures, such as podosomes or focal adhesions (FAs). Since assembly and disassembly of adhesive structures are highly associated with DCs' immunoregulatory and migratory functions, we evaluated how ECTV infection targets podosomes and FAs' organization and formation in natural-host bone marrow-derived DCs (BMDC). We found that ECTV induces a rapid dissolution of podosomes at the early stages of infection, accompanied by the development of larger and wider FAs than in uninfected control cells. At later stages of infection, FAs were predominantly observed in long cellular extensions, formed extensively by infected cells. Dissolution of podosomes in ECTV-infected BMDCs was not associated with maturation and increased 2D cell migration in a wound healing assay; however, accelerated transwell migration of ECTV-infected cells towards supernatants derived from LPS-conditioned BMDCs was observed. We suggest that ECTV-induced changes in the spatial organization of adhesive structures in DCs may alter the adhesiveness/migration of DCs during some conditions, e.g., inflammation.

Objective: Quantify the minimum individual cow colostral immunoglobulin G (IgG) concentration required for pooling to achieve adequate transfer of passive immunity in calves., Animals: 201 Jersey cows., Methods: Colostrum was collected from 28 pools and heat treated before being fed to calves or stored. Parity, total number of cows contributing to the pool, individual cow colostral volume contributions, and total volume of each colostrum pool were recorded. Colostrum IgG concentrations in individual and pooled (pre- and post-heat treatment) samples were analyzed by radial immunodiffusion and Brix refractometry. Colostral IgG concentration of ≥ 50g/L was considered the current recommended dairy industry standard for acceptable colostrum quality. Multivariable models were performed to determine factors affecting pooled colostral IgG concentrations. The minimum colostral IgG concentration required for pooling to achieve the recommended total mass of at least 200g IgG to be fed to a calf was calculated., Results: Total pool volume and the number of cows contributing to the pool were significant factors affecting IgG concentration. Colostrum pools from ≤ 7 cows, with a minimum pool IgG concentration of 70.4 g/L (22.9% Brix) or colostrum pool volume ≤ 40 L, with a minimum pool IgG concentration of 66.2 g/L (21.8% Brix) achieved the recommended total mass of at least 200g IgG in 4L of colostrum., Clinical Relevance: When feeding pooled colostrum, IgG concentrations higher than the industry standard of 50 g/L is recommended to reduce the risk of failure of transfer of passive immunity in calves.

Conventional dendritic cells (cDCs) are innate immune cells that play a pivotal role in inducing antiviral adaptive immune responses due to their extraordinary ability to prime and polarize naïve T cells into different effector T helper (Th) subsets. The two major subpopulations of cDCs, cDC1 (CD8α + in mice and CD141 + in human) and cDC2 (CD11b + in mice and CD1c + in human), can preferentially polarize T cells toward a Th1 and Th2 phenotype, respectively. During infection with ectromelia virus (ECTV), an orthopoxvirus from the Poxviridae family, the timing and activation of an appropriate Th immune response contributes to the resistance (Th1) or susceptibility (Th2) of inbred mouse strains to the lethal form of mousepox. Due to the high plasticity and diverse properties of cDC subpopulations in regulating the quality of a specific immune response, in the present study we compared the ability of splenic cDC1 and cDC2 originating from different ECTV-infected mouse strains to mature, activate, and polarize the Th immune response during mousepox. Our results demonstrated that during early stages of mousepox, both cDC subsets from resistant C57BL/6 and susceptible BALB/c mice were activated upon in vivo ECTV infection. These cells exhibited elevated levels of surface MHC class I and II, and co-stimulatory molecules and showed enhanced potential to produce cytokines. However, both cDC subsets from BALB/c mice displayed a higher maturation status than that of their counterparts from C57BL/6 mice. Despite their higher activation status, cDC1 and cDC2 from susceptible mice produced low amounts of Th1-polarizing cytokines, including IL-12 and IFN-γ, and the ability of these cells to stimulate the proliferation and Th1 polarization of allogeneic CD4 + T cells was severely compromised. In contrast, both cDC subsets from resistant mice produced significant amounts of Th1-polarizing cytokines and demonstrated greater capability in differentiating allogeneic T cells into Th1 cells compared to cDCs from BALB/c mice. Collectively, our results indicate that in the early stages of mousepox, splenic cDC subpopulations from the resistant mouse strain can better elicit a Th1 cell-mediated response than the susceptible strain can, probably contributing to the induction of the protective immune responses necessary for the control of virus dissemination and for survival from ECTV challenge.

The eradication of smallpox was an enormous achievement due to the global vaccination program launched by World Health Organization. The cessation of the vaccination program led to steadily declining herd immunity against smallpox, causing a health emergency of global concern. The smallpox vaccines induced strong, humoral, and cell-mediated immune responses, protecting for decades after immunization, not only against smallpox but also against other zoonotic orthopoxviruses that now represent a significant threat to public health. Here we review the major aspects regarding orthopoxviruses' zoonotic infections, factors responsible for viral transmissions, as well as the emerging problem of the increased number of monkeypox cases recently reported. The development of prophylactic measures against poxvirus infections, especially the current threat caused by the monkeypox virus, requires a profound understanding of poxvirus immunobiology. The utilization of animal and cell line models has provided good insight into host antiviral defenses as well as orthopoxvirus evasion mechanisms. To survive within a host, orthopoxviruses encode a large number of proteins that subvert inflammatory and immune pathways. The circumvention of viral evasion strategies and the enhancement of major host defenses are key in designing novel, safer vaccines, and should become the targets of antiviral therapies in treating poxvirus infections.

Background: It has been proposed that childhood vaccines in high-mortality populations may have substantial impacts on mortality rates that are not explained by the prevention of targeted diseases, nor conversely by typical expected adverse reactions to the vaccines, and that these non-specific effects (NSEs) are generally more pronounced in females. The existence of these effects, and any implications for the development of vaccines and the design of vaccination programs to enhance safety, remain controversial. One area of controversy is the reported association of non-live vaccines with increased female mortality. In a previous randomized controlled trial (RCT), we observed that non-live alum-adjuvanted animal rabies vaccine (ARV) was associated with increased female but not male mortality in young, free-roaming dogs. Conversely, non-live non-adjuvanted human rabies vaccine (NRV) has been associated with beneficial non-specific effects in children. Alum adjuvant has been shown to suppress Th1 responses to pathogens, leading us to hypothesize that alum-adjuvanted rabies vaccine in young dogs has a detrimental effect on female survival by modulating the immune response to infectious and/or parasitic diseases. In this paper, we present the protocol of a 3-arm RCT comparing the effect of alum-adjuvanted rabies vaccine, non-adjuvanted rabies vaccine and placebo on all-cause mortality in an owned, free-roaming dog population, with causal mediation analysis of the RCT and a nested case-control study to test this hypothesis., Methods: Randomised controlled trial with a nested case-control study., Discussion: We expect that, among the placebo group, males will have higher mortality caused by higher pathogen loads and more severe disease, as determined by haematological parameters and inflammatory biomarkers. Among females, we expect that there will be no difference in mortality between the NRV and placebo groups, but that the ARV group will have higher mortality, again mediated by higher pathogen loads and more severe disease. We anticipate that these changes are preceded by shifts in key serum cytokine concentrations towards an anti-inflammatory immune response in females. If confirmed, these results will provide a rational basis for mitigation of detrimental NSEs of non-live vaccines in high-mortality populations., (© 2022. The Author(s).)

The benefits of vaccines have been centred on their specific effects on subsequent infections by target pathogens. Recent studies, however, have opened up new insights into additional effects of vaccines known as non-specific effects (NSEs) or heterologous effects of vaccines. While several articles have reviewed epidemiological and immunological evidence for NSEs of vaccines in humans, similar works on veterinary vaccines are scarce. The objective of this paper was to review the findings of published studies on NSEs of vaccines developed or repurposed for use in animals. In total 8412 titles were retrieved from PubMed and CABI databases on the 30 th of April 2021. After the final stage of screening, 45 eligible articles were included in the review. Data from these articles were summarised and presented here. In general, most of the vaccines studied in the reviewed articles have beneficial NSEs against multiple pathogens and disease conditions. There were, however, fewe studies reporting detrimental NSEs from both non-live and live vaccines which is in contrast to the currently existing evidence of beneficial NSEs of live vaccines and detrimental NSEs of non-live vaccines. This review may be used as a complement for future review of RCT studies of NSEs of vaccines in animals and provide a useful addition to the evolving understanding of the NSEs of vaccines., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Current data strongly suggest herpes simplex virus type 1 (HSV-1) infection in the brain as a contributing factor to Alzheimer's disease (AD). The consequences of HSV-1 brain infection are multilateral, not only are neurons and glial cells damaged, but modifications also occur in their environment, preventing the transmission of signals and fulfillment of homeostatic and immune functions, which can greatly contribute to the development of disease. In this review, we discuss the pathological alterations in the central nervous system (CNS) cells that occur, following HSV-1 infection. We describe the changes in neurons, astrocytes, microglia, and oligodendrocytes related to the production of inflammatory factors, transition of glial cells into a reactive state, oxidative damage, Aβ secretion, tau hyperphosphorylation, apoptosis, and autophagy. Further, HSV-1 infection can affect processes observed during brain aging, and advanced age favors HSV-1 reactivation as well as the entry of the virus into the brain. The host activates pattern recognition receptors (PRRs) for an effective antiviral response during HSV-1 brain infection, which primarily engages type I interferons (IFNs). Future studies regarding the influence of innate immune deficits on AD development, as well as supporting the neuroprotective properties of glial cells, would reveal valuable information on how to harness cytotoxic inflammatory milieu to counter AD initiation and progression.

Dermatophilosis is a form of dermatitis caused by the bacterium Dermatophilus congolensis . The disease usually presents as localized purulent dermatitis, crusty hair masses or widespread matting of the hair. This condition is most common in domestic ruminants; but it can also affect other wild animals and humans. Antimicrobial therapy is used in many regions to treat clinical dermatophilosis with varying results. In this study, we aimed to assess the antimicrobial susceptibility of D. congolensis isolates. Fifty-two isolates were obtained from animals showing clinical signs of the disease at farms in St. Kitts. The isolates were then confirmed as D. congolensis by phenotypic tests, PCR and MALDI-TOF Mass Spectrometry. Furthermore, minimum inhibitory concentrations (MIC) of 16 antimicrobial agents were determined, using the broth microdilution method. Although most antimicrobials showed MICs in line with published values, the tetracycline results displayed a clear bimodal distribution over the tested range, with most isolates showing low MICs and 6 isolates much higher values (+/- 100-fold increase). These results indicate the presence of acquired tetracycline resistance in D. congolensis on the island of St. Kitts. Whether the current observation has implications for efficacy of treating the disease must be confirmed in further research.

Doxycycline (DXC) is a broad-spectrum antibacterial antimicrobial administered to horses for the treatment of bacterial infections which may also affect donkeys. Donkeys have a different metabolism than horses, leading to differences in the pharmacokinetics of drugs compared to horses. This study aimed to describe the population pharmacokinetics of DXC in donkeys. Five doses of DXC hyclate (10 mg/kg) were administered via a nasogastric tube, q12 h, to eight non-fasted, healthy, adult jennies. Serum, urine, synovial fluid and endometrium were collected for 72 h following the first administration. Doxycycline concentration was measured by competitive enzyme immunoassay. Serum concentrations versus time data were fitted simultaneously using the stochastic approximation expectation-maximization algorithm for nonlinear mixed effects. A one-compartment model with linear elimination and first-order absorption after intragastric administration, best described the available pharmacokinetic data. Final parameter estimates indicate that DXC has a high volume of distribution (108 L/kg) as well as high absorption (10.3 h -1 ) in donkeys. However, results suggest that oral DXC at 10 mg/kg q12 h in donkeys would not result in a therapeutic concentration in serum, urine, synovial fluid or endometrium by comparison to the minimum inhibitory concentration of common equine pathogens. Further studies are recommended to identify appropriate dosage and dosing intervals of oral DXC in donkeys.

Dermatophilus congolensis is a bacterial pathogen mostly of ruminant livestock in the tropics/subtropics and certain temperate climate areas. It causes dermatophilosis, a skin disease that threatens food security by lowering animal productivity and compromising animal health and welfare. Since it is a prevalent infection in ruminants, dermatophilosis warrants more research. There is limited understanding of its pathogenicity, and as such, there is no registered vaccine against D. congolensis. To better understanding the genomics of D. congolensis , the primary aim of this work was to investigate this bacterium using whole-genome sequencing and bioinformatic analysis. D. congolensis is a high GC member of the Actinobacteria and encodes approximately 2527 genes. It has an open pan-genome, contains many potential virulence factors, secondary metabolites and encodes at least 23 housekeeping genes associated with antimicrobial susceptibility mechanisms and some isolates have an acquired antimicrobial resistance gene. Our isolates contain a single CRISPR array Cas type IE with classical 8 Cas genes. Although the isolates originate from the same geographical location there is some genomic diversity among them. In conclusion, we present the first detailed genomic study on D. congolensis , including the first observation of tet (Z), a tetracycline resistance-conferring gene.

Dermatophilus congolensis causes dermatophilosis in cattle, mainly in tropical climates. Despite the economic losses caused by this bacterium, its pathogenic factors are less well understood. We report draft genomes of D. congolensis strains isolated during a dermatophilosis outbreak in cattle in St. Kitts and Nevis. Some isolates contain tet (Z), which is responsible for resistance to tetracyclines.

Timely and precise delivery of the endosomal Toll-like receptors (TLRs) to the ligand recognition site is a critical event in mounting an effective antimicrobial immune response, however, the same TLRs should maintain the delicate balance of avoiding recognition of self-nucleic acids. Such sensing is widely known to start from endosomal compartments, but recently enough evidence has accumulated supporting the idea that TLR-mediated signaling pathways originating in the cell membrane may be engaged in various cells due to differential expression and distribution of the endosomal TLRs. Therefore, the presence of endosomal TLRs on the cell surface could benefit the host responses in certain cell types and/or organs. Although not fully understood why, TLR3, TLR7, and TLR9 may occur both in the cell membrane and intracellularly, and it seems that activation of the immune response can be initiated concurrently from these two sites in the cell. Furthermore, various forms of endosomal TLRs may be transported to the cell membrane, indicating that this may be a normal process orchestrated by cysteine proteases-cathepsins. Among the endosomal TLRs, TLR3 belongs to the evolutionary distinct group and engages a different protein adapter in the signaling cascade. The differently glycosylated forms of TLR3 are transported by UNC93B1 to the cell membrane, unlike TLR7, TLR8, and TLR9. The aim of this review is to reconcile various views on the cell surface positioning of endosomal TLRs and add perspective to the implication of such receptor localization on their function, with special attention to TLR3. Cell membrane-localized TLR3, TLR7, and TLR9 may contribute to endosomal TLR-mediated inflammatory signaling pathways. Dissecting this signaling axis may serve to better understand mechanisms influencing endosomal TLR-mediated inflammation, thus determine whether it is a necessity for immune response or simply a circumstantial superfluous duplication, with other consequences on immune response., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Mielcarska, Bossowska-Nowicka and Toka.)

Macrophages are the first encounters of invading bacteria and are responsible for engulfing and digesting pathogens through phagocytosis leading to initiation of the innate inflammatory response. Intracellular digestion occurs through a close relationship between phagocytic/endocytic and lysosomal pathways, in which proteolytic enzymes, such as cathepsins, are involved. The presence of cathepsins in the endo-lysosomal compartment permits direct interaction with and killing of bacteria, and may contribute to processing of bacterial antigens for presentation, an event necessary for the induction of antibacterial adaptive immune response. Therefore, it is not surprising that bacteria can control the expression and proteolytic activity of cathepsins, including their inhibitors - cystatins, to favor their own intracellular survival in macrophages. In this review, we summarize recent developments in defining the role of cathepsins in bacteria-macrophage interaction and describe important strategies engaged by bacteria to manipulate cathepsin expression and activity in macrophages. Particularly, we focus on specific bacterial species due to their clinical relevance to humans and animal health, i.e., Mycobacterium, Mycoplasma , Staphylococcus , Streptococcus, Salmonella , Shigella, Francisella, Chlamydia, Listeria, Brucella, Helicobacter , Neisseria , and other genera., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Szulc-Dąbrowska, Bossowska-Nowicka, Struzik and Toka.)

In susceptible hosts, protection from Leptospira infection is mediated by the innate immune response at the point of entry and humoral immunity. Thus, identifying and segregating the initial host response at the representative host-pathogen interface is needed to understand the typical outcomes of Leptospira infection, clearance, persistence, or disease. An in vitro whole blood culture system to study the overall immune response using pathogenic and non-pathogenic Leptospira strains was explored in this study. Using an ELISA, increased IL-8, TNF alpha, and IL-1 in blood samples stimulated with pathogenic and nonpathogenic Leptospira compared to unstimulated controls were detected. In RT 2 Profiler PCR Array assays, consistent upregulation of 22 genes and downregulation of 25 genes were observed. Few of the notable upregulated genes included BPI, CCL3, CXCL2, IL-6, IL-8, TLR1, TLR2, TLR6, and TNF and downregulated genes included, LBP, LYZ, MPO, MYD88. IFNβ was upregulated in samples treated with pathogenic Leptospira and IL-1β was upregulated in samples treated with nonpathogenic Leptospira. Toll- like Receptor signaling and expression of pattern recognition receptors were two of the five prominent canonical pathways observed. Individual deconvolution of each of the specific and significant pathways observed in this study may improve the understanding of the pathogenesis of this important zoonotic agent. The use of this system in conjunction with whole transcriptome analysis in a larger population, may unveil the robust nature of host/Leptospira interaction., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

TLR3 provides immediate type I IFN response following entry of stimulatory PAMPs into the CNS, as it is in HSV infection. The receptor plays a vital role in astrocytes, contributing to rapid infection sensing and suppression of viral replication, precluding the spread of virus beyond neurons. The route of TLR3 mobilization culminating in the receptor activation remains unexplained. In this research, we investigated the involvement of various types of endosomes in the regulation of the TLR3 mobility in C8-D1A murine astrocyte cell line. TLR3 was transported rapidly to early EEA1-positive endosomes as well as LAMP1-lysosomes following stimulation with the poly(I:C). Later, TLR3 largely associated with late Rab7-positive endosomes. Twenty-four hours after stimulation, TLR3 co-localized with LAMP1 abundantly in lysosomes of astrocytes. TLR3 interacted with poly(I:C) intracellularly from 1 min to 8 h following cell stimulation. We detected TLR3 on the surface of astrocytes indicating constitutive expression, which increased after poly(I:C) stimulation. Our findings contribute to the understanding of cellular modulation of TLR3 trafficking. Detailed analysis of the TLR3 transportation pathway is an important component in disclosing the fate of the receptor in HSV-infected CNS and may help in the search for rationale therapeutics to control the replication of neuropathic viruses., (Copyright © 2020 Mielcarska, Gregorczyk-Zboroch, Szulc-Da̧browska, Bossowska-Nowicka, Wyżewski, Cymerys, Chodkowski, Kiełbik, Godlewski, Gieryńska and Toka.)

Ectromelia virus (ECTV), an orthopoxvirus, undergoes productive replication in conventional dendritic cells (cDCs), resulting in the inhibition of their innate and adaptive immune functions. ECTV replication rate in cDCs is increased due to downregulation of the expression of cathepsins - cystein proteases that orchestrate several steps during DC maturation. Therefore, this study was aimed to determine if downregulation of cathepsins, such as B, L or S, disrupts cDC capacity to induce activating signals in T cells or whether infection of cDCs with ECTV further weakens their functions as antigen-presenting cells. Our results showed that cDCs treated with siRNA against cathepsin B, L and S synthesize similar amounts of pro-inflammatory cytokines and exhibit comparable ability to mature and stimulate alloreactive CD4 + T cells, as untreated wild type (WT) cells. Moreover, ECTV inhibitory effect on cDC innate and adaptive immune functions, observed especially after LPS treatment, was comparable in both cathepsin-silenced and WT cells. Taken together, the absence of cathepsins B, L and S has minimal, if any, impact on the inhibitory effect of ECTV on cDC immune functions. We assume that the virus-mediated inhibition of cathepsin expression in cDCs represents more a survival mechanism than an immune evasion strategy.

Mitochondria are multitasking organelles that play a central role in energy production, survival and primary host defense against viral infections. Therefore, viruses target mitochondria dynamics and functions to benefit their replication and morphogenetic processes. We endeavor to understand the role of mitochondria during infection of ectromelia virus (ECTV), hence our investigations on mitochondria-related genes in non-immune (L929 fibroblasts) and immune (RAW 264.7 macrophages) cells. Our results show that during later stages of infection, ECTV significantly decreases the expression of mitochondria-related genes regulating many aspects of mitochondrial physiology and functions, including mitochondrial transport, small molecule transport, membrane polarization and potential, targeting proteins to mitochondria, inner membrane translocation, and apoptosis. Such down-regulation is cell-specific, since macrophages exhibited a more profound down-regulation of mitochondria-related genes compared to infected L929 fibroblasts. Only L929 cells exhibited up-regulation of two important genes responsible for oxidative phosphorylation and subsequent ATP production: Slc25a23 and Slc25a31. Changes in the expression of mitochondria-related genes are accompanied by altered mitochondria morphology and distribution in both types of cells. In depth Ingenuity Pathway Analysis (IPA) identified the "Sirtuin Signaling Pathway" as the most significant top canonical pathway associated with ECTV infection in both analyzed cell types. Taken together, down-regulation of mitochondria-related genes observed especially in macrophages indicates dysfunctional mitochondria, possibly contributing to energy collapse and induction of intrinsic pathway of apoptosis. Meanwhile, alteration of the expression of several mitochondria-related genes in fibroblasts without apoptosis induction may represent poxviral strategy to control cellular energy metabolism for efficient replication. Keywords: ectromelia virus; mitochondria; fibroblasts; macrophages.

Poxviruses utilize multiple strategies to prevent activation of extrinsic and intrinsic apoptotic pathways for successful replication. Mitochondrial heat shock proteins (mtHsps), especially Hsp60 and its cofactor Hsp10, are engaged in apoptosis regulation; however, until now, the influence of poxviruses on mtHsps has never been studied. We used highly infectious Moscow strain of ectromelia virus (ECTV) to investigate the mitochondrial heat shock response and apoptotic potential in permissive L929 fibroblasts. Our results show that ECTV-infected cells exhibit mostly mitochondrial localization of Hsp60 and Hsp10, and show overexpression of both proteins during later stages of infection. ECTV infection has only moderate effect on the electron transport chain subunit expression. Moreover, increase of mtHsp amounts is accompanied by lack of apoptosis, and confirmed by reduced level of pro-apoptotic Bax protein and elevated levels of anti-apoptotic Bcl-2 and Bcl-xL proteins. Taken together, we show a positive relationship between increased levels of Hsp60 and Hsp10 and decreased apoptotic potential of L929 fibroblasts, and further hypothesize that Hsp60 and/or its cofactor play important roles in maintaining protein homeostasis in mitochondria for promotion of cell survival allowing efficient replication of ECTV.

Background: Cathepsins are a group of endosomal proteases present in many cells including dendritic cells (DCs). The activity of cathepsins is regulated by their endogenous inhibitors - cystatins. Cathepsins are crucial to antigen processing during viral and bacterial infections, and as such are a prerequisite to antigen presentation in the context of major histocompatibility complex class I and II molecules. Due to the involvement of DCs in both innate and adaptive immune responses, and the quest to understand the impact of poxvirus infection on host cells, we investigated the influence of ectromelia virus (ECTV) infection on cathepsin and cystatin levels in murine conventional DCs (cDCs). ECTV is a poxvirus that has evolved many mechanisms to avoid host immune response and is able to replicate productively in DCs., Results: Our results showed that ECTV-infection of JAWS II DCs and primary murine GM-CSF-derived bone marrow cells down-regulated both mRNA and protein of cathepsin B, L and S, and cystatin B and C, particularly during the later stages of infection. Moreover, the activity of cathepsin B, L and S was confirmed to be diminished especially at later stages of infection in JAWS II cells. Consequently, ECTV-infected DCs had diminished ability to endocytose and process a soluble antigen. Close examination of cellular protein distribution showed that beginning from early stages of infection, the remnants of cathepsin L and cystatin B co-localized and partially co-localized with viral replication centers (viral factories), respectively. Moreover, viral yield increased in cDCs treated with siRNA against cathepsin B, L or S and subsequently infected with ECTV., Conclusions: Taken together, our results indicate that infection of cDCs with ECTV suppresses cathepsins and cystatins, and alters their cellular distribution which impairs the cDC function. We propose this as an additional viral strategy to escape immune responses, enabling the virus to replicate effectively in infected cells.

Ectromelia virus (ECTV) is the etiological agent of mousepox, an acute and systemic disease with high mortality rates in susceptible strains of mice. Resistance and susceptibility to mousepox are triggered by the dichotomous T-helper (Th) immune response generated in infected animals, with strong protective Th1 or nonprotective Th2 profile, respectively. Th1/Th2 balance is influenced by dendritic cells (DCs), which were shown to differ in their ability to polarize naïve CD4 + T cells in different mouse strains. Therefore, we have studied the inner-strain differences in the ability of conventional DCs (cDCs), generated from resistant (C57BL/6) and susceptible (BALB/c) mice, to stimulate proliferation and activation of Th cells upon ECTV infection. We found that ECTV infection of GM-CSF-derived bone marrow (GM-BM) cells, composed of cDCs and macrophages, affected initiation of allogeneic CD4 + T cells proliferation in a mouse strain-independent manner. Moreover, infected GM-BM cells from both mouse strains failed to induce and even inhibited the production of Th1 (IFN-γ and IL-2), Th2 (IL-4 and IL-10) and Th17 (IL-17A) cytokines by allogeneic CD4 + T cells. These results indicate that in in vitro conditions ECTV compromises the ability of cDCs to initiate/polarize adaptive antiviral immune response independently of the host strain resistance/susceptibility to lethal infection.

Toll-like receptors (TLRs) sense the presence of pathogen-associated molecular patterns. Nevertheless, the mechanisms modulating TLR-triggered innate immune responses are not yet fully understood. Complex regulatory systems exist to appropriately direct immune responses against foreign or self-nucleic acids, and a critical role of hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), endosomal sorting complex required for transportation-0 (ESCRT-0) subunit, has recently been implicated in the endolysosomal transportation of TLR7 and TLR9. We investigated the involvement of Syk, Hrs, and STAM in the regulation of the TLR3 signaling pathway in a murine astrocyte cell line C8-D1A following cell stimulation with a viral dsRNA mimetic. Our data uncover a relationship between TLR3 and ESCRT-0, point out Syk as dsRNA-activated kinase, and suggest the role for Syk in mediating TLR3 signaling in murine astrocytes. We show molecular events that occur shortly after dsRNA stimulation of astrocytes and result in Syk Tyr-342 phosphorylation. Further, TLR3 undergoes proteolytic processing; the resulting TLR3 N-terminal form interacts with Hrs. The knockdown of Syk and Hrs enhances TLR3-mediated antiviral response in the form of IFN- β , IL-6, and CXCL8 secretion. Understanding the role of Syk and Hrs in TLR3 immune responses is of high importance since activation and precise execution of the TLR3 signaling pathway in the brain seem to be particularly significant in mounting an effective antiviral defense. Infection of the brain with herpes simplex type 1 virus may increase the secretion of amyloid- β by neurons and astrocytes and be a causal factor in degenerative diseases such as Alzheimer's disease. Errors in TLR3 signaling, especially related to the precise regulation of the receptor transportation and degradation, need careful observation as they may disclose foundations to identify novel or sustain known therapeutic targets.

Ectromelia virus (ECTV) is an orthopoxvirus that productively replicates in dendritic cells (DCs), but its influence on the microtubule (MT) cytoskeleton in DCs is not known. Here, we show that ECTV infection of primary murine granulocyte-macrophage colony stimulating factor-derived bone marrow cells (GM-BM) downregulates numerous genes engaged in MT cytoskeleton organization and dynamics. In infected cells, the MT cytoskeleton undergoes dramatic rearrangement and relaxation, accompanied by disappearance of the microtubule organizing centre (MTOC) and increased acetylation and stabilization of MTs, which are exploited by progeny virions for intracellular transport. This indicates a strong ability of ECTV to subvert the MT cytoskeleton of highly specialized immune cells.

Background: Pathogens stimulate immune functions of macrophages. Macrophages are a key sentinel cell regulating the response to pathogenic ligands and orchestrating the direction of the immune response. Our study aimed at investigating the early transcriptomic changes of bovine macrophages (Bomacs) in response to stimulation with CpG DNA or polyI:C, representing bacterial and viral ligands respectively, and performed transcriptomics by RNA sequencing (RNASeq). KEGG, GO and IPA analytical tools were used to reconstruct pathways, networks and to map out molecular and cellular functions of differentially expressed genes (DE) in stimulated cells., Results: A one-way ANOVA analysis of RNASeq data revealed significant differences between the CpG DNA and polyI:C-stimulated Bomac. Of the 13,740 genes mapped to the bovine genome, 2245 had p-value ≤0.05, deemed as DE. At 6 h post stimulation of Bomac, poly(I:C) induced a very different transcriptomic profile from that induced by CpG DNA. Whereas, 347 genes were upregulated and 210 downregulated in response to CpG DNA, poly(I:C) upregulated 761 genes and downregulated 414 genes. The topmost DE genes in poly(I:C)-stimulated cells had thousand-fold changes with highly significant p-values, whereas in CpG DNA stimulated cells had 2-5-fold changes with less stringent p-values. The highest DE genes in both stimulations belonged to the TNF superfamily, TNFSF18 (CpG) and TNFSF10 (poly(I:C)) and in both cases the lowest downregulated gene was CYP1A1. CpG DNA highly induced canonical pathways that are unrelated to immune response in Bomac. CpG DNA influenced expression of genes involved in molecular and cellular functions in free radical scavenging. By contrast, poly(I:C) highly induced exclusively canonical pathways directly related to antiviral immune functions mediated by interferon signalling genes. The transcriptomic profile after poly(I:C)-stimulation was consistent with induction of TLR3 signalling., Conclusion: CpG DNA and poly(I:C) induce different early transcriptional landscapes in Bomac, but each is suited to a specific function of macrophages during interaction with pathogens. Poly(I:C) influenced antiviral response genes, whereas CpG DNA influenced genes important for phagocytic processes. Poly(I:C) was more potent in setting the inflammatory landscape desirable for an efficient immune response against virus infection.

Mitochondria are multifunctional organelles that participate in numerous processes in response to viral infection, but they are also a target for viruses. The aim of this study was to define subcellular events leading to alterations in mitochondrial morphology and function during infection with ectromelia virus (ECTV). We used two different cell lines and a combination of immunofluorescence techniques, confocal and electron microscopy, and flow cytometry to address subcellular changes following infection. Early in infection of L929 fibroblasts and RAW 264.7 macrophages, mitochondria gathered around viral factories. Later, the mitochondrial network became fragmented, forming punctate mitochondria that co-localized with the progeny virions. ECTV-co-localized mitochondria associated with the cytoskeleton components. Mitochondrial membrane potential, mitochondrial fission⁻fusion, mitochondrial mass, and generation of reactive oxygen species (ROS) were severely altered later in ECTV infection leading to damage of mitochondria. These results suggest an important role of mitochondria in supplying energy for virus replication and morphogenesis. Presumably, mitochondria participate in transport of viral particles inside and outside of the cell and/or they are a source of membranes for viral envelope formation. We speculate that the observed changes in the mitochondrial network organization and physiology in ECTV-infected cells provide suitable conditions for viral replication and morphogenesis.

Herpes simplex encephalitis (HSE) is a severe neurological disease in children and adults caused by herpes simplex virus. This review discusses recent findings on the role of Toll-like receptor 3 (TLR3) deficiencies in the HSE development. Critical checkpoints in the TLR3 signaling that contribute to innate response are discussed, including the importance of TLR3 ligand recognition site and transportation in the cell. We also indicate unresolved issues in the TLR3 functioning that might lead to thorough understanding of immunity during HSE. Such a knowledge base will lead to discovery and design of a rationale therapeutic and preventive approach against HSE., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

The aim of the study was to evaluate the influence of ectromelia virus (ECTV) infection on actin cytoskeleton rearrangement in immune cells, such as macrophages and dendritic cells (DCs). Using scanning electron and fluorescence microscopy analysis we observed the presence of long actin-based cellular extensions, formed by both types of immune cells at later stages of infection with ECTV. Such extensions contained straight tubulin filaments and numerous punctuate mitochondria. Moreover, these long cellular projections extended to a certain length and formed convex structures termed "cytoplasmic packets". These structures contained numerous viral particles and presumably were sites of progeny virions' release via budding. Further, discrete mitochondria and separated tubulin filaments that formed a scaffold for accumulated mitochondria were visible within cytoplasmic packets. ECTV-induced long actin-based protrusions resemble "cytoplasmic corridors" and probably participate in virus dissemination. Our data demonstrate the incredible capacity for adaptation of ECTV to its natural host immune cells, in which it can survive, replicate and induce effective mechanisms for viral spread and dissemination., Competing Interests: The authors declare no conflict of interest.

Ectromelia virus (ECTV) belongs to the Orthopoxvirus genus of the Poxviridae family and is a natural pathogen of mice. Certain strains of mice are highly susceptible to ECTV infection and develop mousepox, a lethal disease similar to smallpox of humans caused by variola virus. Currently, the mousepox model is one of the available small animal models for investigating pathogenesis of generalized viral infections. Resistance and susceptibility to ECTV infection in mice are controlled by many genetic factors and are associated with multiple mechanisms of immune response, including preferential polarization of T helper (Th) immune response toward Th1 (protective) or Th2 (non-protective) profile. We hypothesized that viral-induced inhibitory effects on immune properties of conventional dendritic cells (cDCs) are more pronounced in ECTV-susceptible than in resistant mouse strains. To this extent, we confronted the cDCs from resistant (C57BL/6) and susceptible (BALB/c) mice with ECTV, regarding their reactivity and potential to drive T cell responses following infection. Our results showed that in vitro infection of granulocyte-macrophage colony-stimulating factor-derived bone marrow cells (GM-BM-comprised of cDCs and macrophages) from C57BL/6 and BALB/c mice similarly down-regulated multiple genes engaged in DC innate and adaptive immune functions, including antigen uptake, processing and presentation, chemokines and cytokines synthesis, and signal transduction. On the contrary, ECTV infection up-regulated Il10 in GM-BM derived from both strains of mice. Moreover, ECTV similarly inhibited surface expression of major histocompatibility complex and costimulatory molecules on GM-BM, explaining the inability of the cells to attain full maturation after Toll-like receptor (TLR)4 agonist treatment. Additionally, cells from both strains of mice failed to produce cytokines and chemokines engaged in T cell priming and Th1/Th2 polarization after TLR4 stimulation. These data strongly suggest that in vitro modulation of GM-BM innate and adaptive immune functions by ECTV occurs irrespective of whether the mouse strain is susceptible or resistant to infection. Moreover, ECTV limits the GM-BM (including cDCs) capacity to stimulate protective Th1 immune response. We cannot exclude that this may be an important factor in the generation of non-protective Th2 immune response in susceptible BALB/c mice in vivo .

Ectromelia virus (ECTV) is an orthopoxvirus responsible for mousepox, a lethal disease of certain strains of mice that is similar to smallpox in humans, caused by variola virus (VARV). ECTV, similar to VARV, exhibits a narrow host range and has co-evolved with its natural host. Consequently, ECTV employs sophisticated and host-specific strategies to control the immune cells that are important for induction of antiviral immune response. In the present study we investigated the influence of ECTV infection on immune functions of murine GM-CSF-derived bone marrow cells (GM-BM), comprised of conventional dendritic cells (cDCs) and macrophages. Our results showed for the first time that ECTV is able to replicate productively in GM-BM and severely impaired their innate and adaptive immune functions. Infected GM-BM exhibited dramatic changes in morphology and increased apoptosis during the late stages of infection. Moreover, GM-BM cells were unable to uptake and process antigen, reach full maturity and mount a proinflammatory response. Inhibition of cytokine/chemokine response may result from the alteration of nuclear translocation of NF-κB, IRF3 and IRF7 transcription factors and down-regulation of many genes involved in TLR, RLR, NLR and type I IFN signaling pathways. Consequently, GM-BM show inability to stimulate proliferation of purified allogeneic CD4+ T cells in a primary mixed leukocyte reaction (MLR). Taken together, our data clearly indicate that ECTV induces immunosuppressive mechanisms in GM-BM leading to their functional paralysis, thus compromising their ability to initiate downstream T-cell activation events.

Poxviruses have evolved numerous mechanisms to avoid the immune response of the infected host, and many of these mechanisms have not been fully described. Here, we studied the transcriptional response of innate immune genes in BALB/c and C57BL/6 peritoneal macrophages following infection with the Moscow strain of ectromelia virus (ECTV-Mos) with the aim of delineating innate immune genes that contribute to the difference between susceptibility and resistance to lethal infection. We show a generalized downregulation of many genes in four categories (toll-like receptor signaling, NOD-like receptor signaling, RIG-I-like receptor signaling, and type I interferon signaling) of antiviral innate immune receptors, downstream signaling pathways, and responsive components. Two important observations were made. First, 14 innate antiviral genes were differentially expressed with fold change upregulation of two and above occurring in C57BL/6 mice, known to be resistant to ECTV-Mos infection, whereas the same genes were downregulated in BALB/c mice with fold change of two and below. Second, the cathepsin group of genes was downregulated in both strains of mice but with profound fold changes of 17, 38, and 62 downregulation for CtsL, CtsB, and CtsS, respectively, in C57BL/6 mice. We show that a poxvirus profoundly downregulates both the mRNA and protein expression of these three cathepsins and this change appears to support virus replication. Based on these data we propose that the variations in gene expression observed may contribute to the difference in resistance/susceptibility between BALB/c and C57BL/6 mice to lethal infection by ECTV-Mos.

Ectromelia virus (ECTV, the causative agent of mousepox), which represents the same genus as variola virus (VARV, the agent responsible for smallpox in humans), has served for years as a model virus for studying mechanisms of poxvirus-induced disease. Despite increasing knowledge on the interaction between ECTV and its natural host-the mouse-surprisingly, still little is known about the cell biology of ECTV infection. Because pathogen interaction with the cytoskeleton is still a growing area of research in the virus-host cell interplay, the aim of the present study was to evaluate the consequences of ECTV infection on the cytoskeleton in a murine fibroblast cell line. The viral effect on the cytoskeleton was reflected by changes in migration of the cells and rearrangement of the architecture of tubulin, vimentin, and actin filaments. The virus-induced cytoskeletal rearrangements observed in these studies contributed to the efficient cell-to-cell spread of infection, which is an important feature of ECTV virulence. Additionally, during later stages of infection L929 cells produced two main types of actin-based cellular protrusions: short (actin tails and "dendrites") and long (cytoplasmic corridors). Due to diversity of filopodial extensions induced by the virus, we suggest that ECTV represents a valuable new model for studying processes and pathways that regulate the formation of cytoskeleton-based cellular structures. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Th17 cells are a relatively newly discovered subpopulation of helper CD4+ T lymphocytes. It has been shown that these cells may contribute to tissue damage during certain inflammatory and autoimmune diseases and also play an important role in antitumor and antimicrobial, particularly antibacterial, immunity. Bacteria stimulate the Th17 response through several Toll-like (TLR), NOD-like (NLR) and C-type lectin (CLR) receptors. When activated, Th17 lymphocytes produce several cytokines, mainly interleukin (IL)-17 and chemokines, that further attract and activate phagocytes to mediate bacterial clearance. Thus Th17 cells contribute to induction of host protective immunity, particularly against extracellular bacterial pathogens: Staphylococcus aureus, Streptococcus pneumoniae and Klebsiella pneumoniae. Furthermore, numerous studies indicate the importance of Th17 lymphocytes in immunity against intracellular bacteria such as Francisella tularensis and Chlamydia muridarum. In this case, the protective immune response is mediated mainly through stimulation of local dendritic cell (DC) function for establishing a Th1 immune response, indispensable for controlling intracellular infectious agents. However, deregulation of the Th17/IL17 response during bacterial infections may lead to profound pathologies. As a result, Th17 cells participate in chronic inflammatory diseases, leading to tissue destruction and favoring tumor development. This article summarizes current understanding of the bacteriainduced Th17 response in the context of the protective immune response and immunopathology.

Natural killer (NK) cells play a role in innate antiviral immunity by directly lysing virus-infected cells and producing antiviral cytokines such as interferon gamma (IFN-γ). We developed a system for characterizing the bovine NK response to foot-and-mouth disease virus (FMDV), which causes a disease of cloven-hoofed animals and remains a threat to livestock industries throughout the world. IL-2 stimulation of PBMC resulted in poor killing of human K562 cells, which are often used as NK target cells, while lysis of the bovine BL3.1 cell line was readily detected. Depletion of NKp46-expressing cells revealed that 80% of the killing induced by IL-2 could be attributed to NKp46(+) cells. In order to characterize the response of NK cells against FMDV in vivo, we infected groups of cattle with three different strains of the virus (A24 Cruzeiro, O1 Manisa, O Hong Kong) and evaluated the cytolytic ability of NK cells through the course of infection. We consistently observed a transient increase in cytolysis, although there was variation in magnitude and kinetics. This increase in cytolysis remained when CD3(+) cells were removed from the preparation of lymphocytes, indicating that cytolysis was independent of MHC-T cell receptor interaction or γδ T cell activation. In contrast, animals monitored following vaccination against FMDV did not exhibit any increase in NK killing. These data suggest that NK cells play a role in the host immune response of cattle against FMDV, and contrast with the suppression of NK activity previously observed in swine infected with FMDV., (Published by Elsevier Ltd.)

Pathogens in general and pathogenic viruses in particular have evolved a myriad of mechanisms to escape the immune response of mammalian species. Viruses that cause acute disease tend to bear characteristics that make them very contagious, as survival does not derive from chronicity of infection, but spread of disease throughout the herd. Foot-and-mouth disease virus (FMDV) is one of the most contagious viruses known. Upon infection of susceptible species, cloven-hoofed animals, the virus proliferates rapidly and causes a vesicular disease within 2-4 days. Disease symptoms resolve by 10 days to 2 weeks and in most cases, virus can no longer be detected. Periods of fever and viremia are usually brief, 1-3 days. In vivo control of virus infection and clearance of the virus during and following acute infection is of particular interest. The interaction of this virus with cells mediating the early, innate immune response has been analyzed in a number of recent studies. In most reports, the virus has a distinct inhibitory effect on the response of cells early in infection. Here we review these new data and discuss the dynamics of the interaction of virus with different cell types mediating the immune response to infection., (Copyright © 2013 Elsevier B.V. All rights reserved.)

γδ T cells are the majority peripheral blood T cells in young cattle. The role of γδ T cells in innate responses against infection with foot-and-mouth disease virus was analyzed on consecutive 5 d following infection. Before infection, bovine WC1(+) γδ T cells expressed a nonactivated phenotype relative to CD62L, CD45RO, and CD25 expression and did not produce IFN-γ ex vivo. Additionally, CD335 expression was lacking and no spontaneous target cell lysis could be detected in vitro, although perforin was detectable at a very low level. MHC class II and CD13 expression were also lacking. Following infection with foot-and-mouth disease virus, expression of CD62L and CD45RO was greatly reduced on WC1(+) γδ T cells, and unexpectedly, CD45RO expression did not recover. A transient increase in expression of CD25 correlated with production of IFN-γ. Expression of CD335 and production of perforin were detected on a subset of γδ T cells, and this correlated with an increased spontaneous killing of xenogeneic target cells. Furthermore, increased MHC class II expression was detected on WC1(+) γδ T cells, and these cells processed protein Ags. These activities are rapidly induced, within 3 d, and wane by 5 d following infection. All of these functions, NK-like killing, Ag processing, and IFN-γ production, have been demonstrated for these cells in various species. However, these results are unique in that all these functions are detected in the same samples of WC1(+) γδ T cells, suggesting a pivotal role of these cells in controlling virus infection.

Foot-and-mouth disease (FMD) continues to be a significant threat to the health and economic value of livestock species. This acute infection is caused by the highly contagious FMD virus (FMDV), which infects cloven-hoofed animals, including large and small ruminants and swine. Current vaccine strategies are all directed toward the induction of neutralizing antibody responses. However, the role of cytotoxic T lymphocytes (CTLs) has not received a great deal of attention, in part because of the technical difficulties associated with establishing a reliable assay of cell killing for this highly cytopathic virus. Here, we have used recombinant human adenovirus vectors as a means of delivering FMDV antigens in a T cell-directed vaccine in pigs. We tested the hypothesis that impaired processing of the FMDV capsid would enhance cytolytic activity, presumably by targeting all proteins for degradation and effectively increasing the class I major histocompatibility complex (MHC)/FMDV peptide concentration for stimulation of a CTL response. We compared such a T cell-targeting vaccine with the parental vaccine, previously shown to effectively induce a neutralizing antibody response. Our results show induction of FMDV-specific CD8(+) CTL killing of MHC-matched target cells in an antigen-specific manner. Further, we confirm these results by MHC tetramer staining. This work presents the first demonstration of FMDV-specific CTL killing and confirmation by MHC tetramer staining in response to vaccination against FMDV.

Foot-and-mouth disease virus (FMDV) causes an acute, highly contagious disease of livestock. Though FMDV is very sensitive to interferon-alpha (IFN-alpha), IFN-beta, and IFN-gamma, the virus has evolved mechanisms to evade such innate responses. For instance, during acute infection, FMDV suppresses IFN-alpha production by skin and myeloid dendritic cells (DCs). We have previously reported that FMDV infection induces a transient lymphopenia and interruption of T-lymphocyte responses to mitogenic stimuli. To further understand the immunopathogenesis of FMD, we have now analyzed the serum IFN-alpha response in relation to lymphopenia, and the number and function of plasmacytoid DCs (pDCs) following infection of pigs with multiple serotypes of FMDV. Serum IFN-alpha peaked 2-3 d post-infection (PI), regardless of FMDV serotype. Lymphopenia coincided with peak viremia and the serum IFN-alpha response. Circulating pDC numbers and in-vitro pDC IFN-alpha secretion transiently declined by 48 h following infection. Infection of lymphocytes or pDCs was never detected regardless of the FMDV serotype inoculated or the age of the animal infected. These data indicate that, like other DC subsets, there is suppression of interferon production by pDCs, which abrogates this important innate response. Rapid induction of serum IFN-alpha, albeit short-lived, may contribute to the rapid resolution of FMDV viremia prior to induction of specific immunity.

Natural killer (NK) cells provide one of the initial barriers of cellular host defense against pathogens, in particular intracellular pathogens. The role of these cells in foot-and-mouth disease virus (FMDV) infection is unknown. Previously, we characterized the phenotype and function of NK cells from swine (F. N. Toka et al., J. Interferon Cytokine Res. 29:179-192, 2009). In the present study, we report the analysis of NK cells isolated from animals infected with FMDV and tested ex vivo and show that NK-dependent cytotoxic activity against tumor cells as targets was impaired. More relevantly to this infection, the killing of target cells infected with FMDV also was inhibited. Further, the proportion of NK cells capable of producing gamma interferon and storing perforin was reduced. Peripheral blood mononuclear cells isolated from infected animals are not productively infected, but virus exposure in vivo resulted in the significant induction of NKp30 and Toll-like receptor 3 expression and the moderate activation of SOCS3 and interleukin-15 receptor mRNA. However, there was little alteration of mRNA expression from a number of other receptor genes in these cells, including SH2D1B and NKG2A (inhibitory) as well as NKp80, NKp46, and NKG2D (activating). These data indicate that this virus infection influences the ability of NK cells to recognize and eliminate FMDV-infected cells. In addition, a reduction in NK cell cytotoxicity coincided with the increase in virus titers, indicating the virus blocking of NK cell-associated innate responses, albeit temporarily. These effects likely culminate in brief but effective viral immune evasion, allowing the virus to replicate and disseminate within the host.

The induction of innate immune responses by toll-like receptor (TLR) agonists is the subject of intense investigation. In large part, this reflects the potential of such compounds to be effective vaccine adjuvants. For that reason, we analyzed the activation of innate cells in swine by TLR7 and TLR8 agonists. These agonists activated porcine NK cells by increasing gamma interferon (IFN-gamma) expression and perforin storage. The activation of porcine NK cells was mediated by accessory cells, since their depletion resulted in reduced cytotoxicity toward target cells. Accessory cells were stimulated to produce interleukin 12 (IL-12), IL-15, IL-18, and IFN-alpha after treatment with TLR7 or TLR8 agonists. Neutralization of these cytokines reduced but did not completely inhibit the induction of NK cell cytotoxicity. Direct stimulation of NK cells with TLR7 or TLR8 agonists resulted in minimal cytotoxicity but levels of IFN-gamma equivalent to those detected in the presence of accessory cells. Porcine NK cells express both TLR7 and TLR8 mRNAs, and treatment with these TLR agonists induced higher mRNA expression levels of TRAIL and IL-15Ralpha, which may contribute to the activity of NK cells. These data indicate that TLR7 and TLR8 agonists indirectly or directly activate porcine NK cells but that optimum levels of activation require cytokine secretion by accessory cells activated by these compounds. Interestingly, NK cells activated by TLR7 or TLR8 agonists were cytotoxic against foot-and-mouth disease virus (FMDV)-infected cells in vitro, indicating that these TLR agonists may be beneficial as adjuvants to stimulate the innate immunity against FMDV.

Natural killer (NK) cells play a vital role in innate response against viral infections and cellular transformation. In vivo modulation of their response may enhance their antiviral function. Here we describe the phenotype of porcine NK cells, test potential proinflammatory cytokines for activation of these cells and assess the capability of porcine NK cells to kill virus-infected or tumor cells in vitro. The CD2+/CD8+/CD3(-) cell compartment contained porcine NK cells, which at the resting stage were minimally cytotoxic toward foot-and-mouth disease virus (FMDV)-infected porcine cells or tumor cell lines. Direct stimulation of NK cells with proinflammatory cytokines induced efficient lysis of FMDV-infected cells with interleukin (IL)-2 or IL-15 showing the highest stimulatory capacity. Lower levels of NK cell activation were induced by IL-12, IL-18, or interferon (IFN)-alpha, however, IL-12 and IL-18 synergistically activated NK cells. Combinations of IL-15 and IL-12 or IL-15 and IL-18 did not further increase the porcine NK cell lytic capability over IL-15 alone. Natural killer cells expressed IFN-gamma regardless of the cytokine used for stimulation while expression of perforin increased modestly. The enhancement of porcine NK cell activity by proinflammatory cytokines offers a promising tool for development of antiviral approaches against virus infection.

Langerhans cells (LCs) are resident dendritic cells (DCs) of skin and mucosal epithelium. The standard for identifying skin DCs as LCs is expression of langerin (CD207), a surface protein that mediates Birbeck granule (BG) formation upon internalization. Reports of BGs in porcine skin DC are contradictory, due to lack of langerin detection. Here, we present the sequence of porcine langerin/CD207, showing that the predicted porcine protein shares 75%/86% amino acid identity/similarity with human. Langerin mRNA was detected in porcine skin DCs by PCR and langerin protein was detected in both isolated skin DCs and skin sections by immunostaining. Approximately, 50-70% of skin DCs expressed langerin, demonstrating that the majority of porcine skin DCs are LCs. The full length sequence combined with the identification of antibodies reactive with porcine langerin, facilitates the study of LCs in swine, and advances the use of swine for studying skin diseases and infectious disease processes involving skin.

Summary: The interface between successful pathogens and their hosts is often a tenuous balance. In acute viral infections, this balance involves induction and inhibition of innate responses. Foot-and-mouth disease virus (FMDV) is considered one of the most contagious viruses known and is characterized by rapid induction of clinical disease in cloven hoofed animals exposed to infection. Viral shedding is extensive before the equally rapid resolution of acute disease. This positive strand RNA virus is an extremely successful pathogen, due in part to the ability to interrupt the innate immune response. Previous reviews have described the inhibition of cellular innate responses in the infected cell both in vitro and in vivo. Here, we present a review of virus inhibition of cells that are a source of antiviral function in swine. Particularly in the case of dendritic cells and natural killer cells, the virus has evolved mechanisms to interrupt the normal function of these important mediators of innate function, even though these cells are not infected by the virus. Understanding how this virus subverts the innate response will provide valuable information for the development of rapidly acting biotherapeutics to use in response to an outbreak of FMDV.

Viruses have evolved multiple mechanisms to evade the innate immune response, particularly the actions of interferons (IFNs). We have previously reported that exposure of dendritic cells (DCs) to foot-and-mouth disease virus (FMDV) in vitro yields no infection and induces a strong type I IFN (IFN-alpha and IFN-beta) response, indicating that DCs may play a critical role in the innate response to the virus. In vivo, FMDV induces lymphopenia and reduced T-cell proliferative responses to mitogen, viral effects that may contribute to evasion of early immune responses. In this study we analyzed the in vivo effects of FMDV infection on the IFN-alpha response of two populations of dendritic cells. During the acute phase of infection of swine, production of IFN-alpha from monocyte-derived DCs (MoDCs) and skin-derived DCs (skin DCs) is inhibited. This effect occurs concurrently with rising viral titers in the blood; however, these cells are not productively infected. Interestingly, there are no changes in the capability of these DCs to take up particles and process antigens, indicating that antigen-presenting cell function is normal. These data indicate that inhibition of the IFN-alpha response of dendritic cell populations from blood and skin by FMDV enhances viral pathogenesis in infected animals.

In a DNA immunization against Herpes simplex virus (HSV), we examined the ability of plasmid-encoded interleukin-15 (pIL-15) to induce and maintain the mucosal B and T cell immune response. pIL-15 generated memory CD8(+) T cell responses that were threefold higher and mainly maintained in the spleen, but high levels of immunoglobulin A antibodies were induced and maintained long-term in the vaginal mucosa. Both of these enhanced components of the immune responses were recalled rapidly upon challenge with a lethal dose of HSV McKrae, affording protection in mice immunized with codelivery of pIL-15. Our results show for the first time that intranasal administration of pIL-15 along with plasmid-encoded glycoprotein B of HSV leads to enhancement of primary and memory CD8(+) T cell responses as well as humoral immune response. Therefore, a mucosal immunization strategy that incorporates a potent cytokine such as IL-15 as an adjuvant might induce protective mucosal immune responses that constitute the initial barrier at mucosal portals of pathogen entry.

The capability of cellular immune components to rapidly recall upon challenge in most situations decides the efficacy of a vaccine. Here, we show that immunization of mice with SSIEFARL peptide (immunodominant epitope in glycoprotein B of herpes simplex virus type 1, aa498-505) combined with TLR9 ligand in the absence of helper CD4(+) T cell activation generates a functionally impaired CD8(+) T cell memory response. Codelivery of IL-12, IL-15, or anti-CD40 together with MHC class-I-restricted peptide combined with TLR9 ligand at inception of immunization resulted in generation of memory CD8(+) T cells that were several fold less compromised than immunization with peptide alone. Furthermore, administration of either plasmid DNA encoding IL-15 or anti-CD40 mAb but not rIL-12 during the memory phase restored the reactivity of memory CD8(+) T cells. Moreover, the rescued CD8(+) T cells preserved their cytotoxic capability and were able to clear a recombinant vaccinia virus encoding glycoprotein B of HSV. Our results indicate that good memory CD8(+) T cell response to peptide immunization can be achieved by using costimulatory procedures at the time of priming or recall immunization.

It has become evident that naturally occurring CD25(+) regulatory T cells (T(reg) cells) not only influence self-antigen specific immune response but also dampen foreign antigen specific immunity. This report extends our previous findings by demonstrating that immunity to certain herpes simplex virus (HSV) vaccines is significantly elevated and more effective if T(reg) cell response is curtailed during either primary or recall immunization. The data presented here show that removal of CD25(+) T(reg) cells prior to SSIEFARL-CpG or gB-DNA immunization significantly enhanced the resultant CD8(+) T-cell response to the immunodominant SSIEFARL peptide. The enhanced CD8(+) T-cell reactivity in T(reg) cell-depleted animals was between two- and threefold and evident in both acute and memory stages. Interestingly, removal of CD25(+) T(reg) cells during the memory recall response to plasmid immunization resulted in a twofold increase in CD8(+) T-cell memory pool. Moreover, in the challenge experiments, memory CD8(+) T cells generated with plasmid DNA in the absence of T(reg) cells cleared the virus more effectively compared with control groups. We conclude that CD25(+) T(reg) cells quantitatively as well as qualitatively affect the memory CD8(+) T-cell response generated by gB-DNA vaccination against HSV. However, it remains to be seen if all types of vaccines against HSV are similarly affected by CD25(+) T(reg) cells and if it is possible to devise means of limiting T(reg) cell activity to enhance vaccine efficacy.

Mucosal surfaces represent the entry route of a multitude of viral pathogens. For many of these viruses, such as the herpes simplex viruses and human immunodeficiency virus, no effective vaccine exists. Hence, it is important that prospective vaccines engender maximal immunity at these susceptible sites. Genetic vaccines encoding adjuvant molecules represent one approach to optimize mucosal as well as systemic immunity. Promising candidates include various inflammatory cytokines and chemokines that might be used to enhance the primary response to a level sufficient for protection. Encouraging studies involving cytokines such as granulocyte/macrophage colony-stimulating factor, interleukin-2 (IL-2), IL-12, IL-18, and many others are examined. Notable chemokines that may offer hope in such efforts include IL-8, RANTES, CCL19, CCL21, and a few others. Combinatorial approaches utilizing several cytokines and chemokines will most likely yield the greatest success. In addition, as more is discovered regarding the requirements for memory development of T cells, boosters involving key cytokines such as IL-15 and IL-23 may prove beneficial to long-term maintenance of the memory pool. This review summarizes the progress in the use of genetic vaccines to achieve mucosal immunity and discusses the needed strategies to maximize long-term prospective immunity at this vulnerable entry site.

Humoral and cellular immunity, associated with long-term protective immunological memory, defines the efficacy of a given vaccine formulation. However, few vaccines achieve this target without the aid of a suitable adjuvant. Molecular adjuvants in vaccination against infectious agents offer a noninvasive means of enhancing the immune response against target antigens. To examine the potency of two beta-chemokines as immunomodulators, plasmid DNA encoding beta-chemokines CCL19 and CCL21 (CCR7L) was codelivered intranasally with plasmid DNA or recombinant vaccinia virus encoding herpes simplex virus (HSV) gB (HSV-gB) in a prime-and-boost vaccination strategy. This vaccination regimen increased serum and vaginal immunoglobulin G (IgG) and IgA, respectively, as well as the numbers of HSV-gB(498-505) peptide-specific gamma interferon-producing CD8(+) T cells. Distinctively, a high number of cytotoxic T lymphocytes was achieved when pCCR7L was applied at both prime and boost as opposed to omission of pCCR7L. A rapid-recall response was induced in the genital tract upon challenge with the HSV McKrae strain, affording a high level of protection and survival of vaccinated mice. Our results demonstrate that high innate immune kinetics and distribution of adaptive response induced in the nasal mucosa appears to be key factors in generating protective memory responses against HSV. Thus CCR7L expressed ectopically may serve as a molecular adjuvant to boost the immune response to a codelivered antigen in mucosal surfaces.

Genetically sensitive mice (i.e. H-2(d) haplotype) infected with a natural mouse pathogen named ectromelia virus (EV) can develop a mousepox. Virus replicates well in the skin, next in the draining lymph nodes (DLNs) and then in the spleen and liver, where it may induce extensive necrosis with strong inflammatory reaction. It is well known from the studies defined on some other viruses that a correlation, functional link and powerful help exist between MHC class I-restricted CD8(+) and MHC class II-restricted CD4(+) virus-specific cytotoxic T lymphocytes (CTLs). However, in the case of mousepox the role of CD4(+) CTLs is still controversial and some reports support the notion that induction of EV-specific CD4(+) CTLs is nonessential for the generation of virus-specific immune response. Consequently, this study was designed to evaluate EV-specific CD8(+) and CD4(+) CTL activity in the DLNs, spleen, skin and conjunctivae of BALB/c (H-2(d)) mice at 7 and 14 days p.i. with Moscow strain of EV. By using bulk cytotoxicity assay and immunosurgery of effector T cells with mAb specific for CD4(+) and/or CD8(+) T cells our data show that EV-specific CD8(+) CTLs predominated in DLNs and spleen at 7 days (67 and 66% of total CTLs, respectively) and 14 days p.i. (63 and 69% of total CTLs, respectively). In contrast, we found that EV clearance from the cutaneous lesions during mousepox is CD4(+) CTL-dependent at 7 days p.i. (59% of total CTLs), whereas at 14 days p.i. CD8(+) CTLs predominated in the epidermis, accounting for 72% of the total EV-specific CTLs. Our studies showed that the population of EV-specific CTLs is heterogeneous and contains cells of both phenotypes: CD8(+) and CD4(+). However, these effector cells did not express a similar tendency in cytotoxic activity in the DLNs, spleen and skin in comparison to the conjunctivae where EV-specific CD8(+) and CD4(+) CTLs were not detected at 7 days p.i. and at peak of mousepox conjunctivitis (14 days p.i.). Our results are discussed in terms of the value of EV to study antiviral CTL responses in the genetically susceptible host.