Epithelial distribution and replication of foot-and-mouth disease virus RNA in infected pigs

Although the pathogenesis of foot-and-mouth disease (FMD) has been extensively investigated, relatively few studies have addressed the localization of FMD virus (FMDV) and in particular its replication in relation to the typical in-vivo sites of FMD lesions. In the present study, pigs were infected experimentally with FMDV serotype O UKG 34/2001 and tissue samples were collected from 1 to 4 days post-infection. Samples were stored at -70 degrees C and frozen sections were prepared for in-situ hybridization (ISH). A digoxigenin-labelled RNA probe complementary to a coding part of the RNA-dependent RNA polymerase (3D) genomic region was prepared. The FMDV positive strand RNA was prominent in the basal layers of the epithelium. A diffuse positive signal was also noted in the cytoplasm of cells of the stratum spinosum of lesional epithelium, but there was no signal in the stratum corneum. Detection of FMDV negative strand RNA was observed in basal cells above the basement membrane and along the dermal papillae. The basal cells therefore demonstrate the highest signal for detection of the FMDV positive and negative strand RNAs in both tongue and foot epithelium. These novel results suggest that the epithelial basal cells could be an early replication site of FMDV in vivo.     

Cross-inhibition to heterologous foot-and-mouth disease virus infection induced by RNA interference targeting the conserved regions of viral genome

RNA interference (RNAi) is the process by which double-stranded RNA (dsRNA) directs sequence-specific degradation of messenger RNA in animal and plant cells. In mammalian cells, RNAi can be triggered by 21-23 nucleotide duplexes of small interfering RNA (siRNA). Strategies to inhibit RNA virus multiplication based on the use of siRNAs have to consider the high genetic polymorphism exhibited by this group of virus. Here we described a significant cross-inhibition of foot-and-mouth disease (FMD) virus (FMDV) replication in BHK-21 cells by siRNAs targeted to various conserved regions (5'NCR, VP4, VPg, POL, and 3'NCR) of the viral genome. The results showed that siRNAs generated in vitro by human recombinant dicer enzyme gave an inhibition of 10- to 1000-fold in virus yield of both homologous (HKN/2002) and heterologous (CHA/99) isolates of FMDV serotype O at 48 h post-infection (hpi). The inhibition extended to at least 6 days post-infection. For serotype Asia1, the virus yield in YNBS/58-infected cells examined at 12, 24, and 48 hpi decreased by approximately 10-fold in cells pretreated with HKN/2002-specific siRNAs, but there was no significant decrease at 60 hpi. The inhibition was specific to FMDV replication, as no reduction was observed in virus yield of pseudorabies virus, an unrelated virus. Moreover, we also demonstrated an enhanced viral suppression could be achieved in BHK-21 cells with siRNA transfection after an infection had been established. These results suggested that siRNAs directed to several conserved regions of the FMDV genome could inhibit FMDV replication in a cross-resistance manner, providing a strategy candidate to treat high genetic variability of FMDV.     

Foot-and-mouth disease virus utilizes an autophagic pathway during viral replication

Foot-and-mouth disease virus (FMDV) is the type species of the Aphthovirus genus within the Picornaviridae family. Infection of cells with positive-strand RNA viruses results in a rearrangement of intracellular membranes into viral replication complexes. The origin of these membranes remains unknown; however induction of the cellular process of autophagy is beneficial for the replication of poliovirus, suggesting that it might be advantageous for other picornaviruses. By using confocal microscopy we showed in FMDV-infected cells co-localization of non-structural viral proteins 2B, 2C and 3A with LC3 (an autophagosome marker) and viral structural protein VP1 with Atg5 (autophagy-related protein), and LC3 with LAMP-1. Importantly, treatment of FMDV-infected cell with autophagy inducer rapamycin, increased viral yield, and inhibition of autophagosomal pathway by 3-methyladenine or small-interfering RNAs, decreased viral replication. Altogether, these studies strongly suggest that autophagy may play an important role during the replication of FMDV.     

Evidence for a role of Langerhans cell-derived IL-16 in atopic dermatitis

BACKGROUND: The factors controlling infiltration of inflammatory cells into atopic dermatitis (AD) lesions remain to be fully explored. Recently, epidermal cells in lesional AD were reported to contain increased messenger (m)RNA levels of IL-16, a cytokine that induces chemotactic responses in CD4(+)T cells, monocytes, and eosinophils. OBJECTIVES: We sought to determine the expression of IL-16 in epidermal cells in normal skin and skin from AD lesions and to investigate whether Langerhans cell (LC)-derived IL-16 may contribute to the initiation of atopic eczema. METHODS: The cutaneous expression of IL-16 was investigated by in situ hybridization and immunohistochemistry. Expression of IL-16 was also investigated in freshly isolated LCs and in keratinocytes by intracellular cytokine staining, quantitative real-time RT-PCR, and ELISA. RESULTS: Low levels of IL-16 mRNA, but no stored IL-16 protein, were detected in keratinocytes and LCs isolated from normal skin. Synthesis, storage, and secretion of IL-16 could be induced in LCs, but not keratinocytes, by activation with phorbol ester and ionomycin. In normal skin (n = 10) neither keratinocytes nor LCs expressed IL-16. In contrast, IL-16 was contained in approximately 40% of CD1a(+)LCs in patients with active AD (n = 16). IL-16 expression in LCs in patients with AD correlated with the number of infiltrating CD4(+)cells (r =.72, P =.0017) and was completely downregulated parallel to the clinical response of AD lesions to topical treatment with FK506. CONCLUSION: LC-derived IL-16 may participate in the recruitment and activation of inflammatory cells in AD.     

Susceptibility to viral infection is enhanced by stable expression of 3A or 3AB proteins from foot-and-mouth disease virus

The foot-and-mouth disease virus (FMDV) 3A protein is involved in virulence and host range. A distinguishing feature of FMDV 3B among picornaviruses is that three non-identical copies are encoded in the viral RNA and required for optimal replication in cell culture. Here, we have studied the involvement of the 3AB region on viral infection using constitutive and transient expression systems. BHK-21 stably transformed clones expressed low levels of FMDV 3A or 3A(B) proteins in the cell cytoplasm. Transformed cells stably expressing these proteins did not exhibit inner cellular rearrangements detectable by electron microscope analysis. Upon FMDV infection, clones expressing either 3A alone or 3A(B) proteins showed a significant increase in the percentage of infected cells, the number of plaque forming units and the virus yield. The 3A-enhancing effect was specific for FMDV as no increase in viral multiplication was observed in transformed clones infected with another picornavirus, encephalomyocarditis virus, or the negative-strand RNA virus vesicular stomatitis virus. A potential role of 3A protein in viral RNA translation was discarded by the lack of effect on FMDV IRES-dependent translation. Increased viral susceptibility was not caused by a released factor; neither the supernatant of transformed clones nor the addition of purified 3A protein to the infection medium was responsible for this effect. Unlike stable expression, high levels of 3A or 3A(B) protein transient expression led to unspecific inhibition of viral infection. Therefore, the effect observed on viral yield, which inversely correlated with the intracellular levels of 3A protein, suggests a transacting role operating on the FMDV multiplication cycle.     

The effects of gamma interferon on replication of foot-and-mouth disease virus in persistently infected bovine cells

Summary.  Foot-and-mouth disease virus (FMDV) causes a highly contagious viral disease of cloven-hoofed animals, which has a considerable socio-economic impact on the countries affected. In addition, persistent infection can occur following clinical or sub-clinical disease in either vaccinated or non-vaccinated cattle. The mechanism(s) by which FMDV persistence is established and maintained is not fully understood. To better understand the basic mechanisms controlling the virus infection in cattle, the effects of interferon gamma (IFN-γ) on the replication of FMDV was evaluated in vitro in persistently infected-epithelial cells isolated from FMDV infected cattle. Initially primary bovine thyroid (BTY) cells were treated with varying doses of bovine recombinant IFN-γ. The cytokine activity was measured by detection of viral antigen in cell supernatants and viral RNA expression compared with cells without INF-γ treatment. Pretreatment with IFN-γ profoundly affected FMDV growth in BTY cells. The replication of FMDV was affected in the presence of more than 2.5 u/ml of IFN-γ and the effect was both dose-dependent and related to the time of exposure. Analysis of the mechanism of inhibition suggests that IFN-γ did not inhibit the viral replication through induction of nitric oxide. More interesting is the finding that continuous treatment with IFN-γ severely restricts FMDV replication or even cures persistently infected bovine epithelial cells, indicating that a cytokine-mediated pathway may be involved in the in vivo clearance of persistent FMDV. Received June 27, 2001; accepted June 11, 2002     

Evidence that the GBV-C/hepatitis G virus is primarily a lymphotropic virus

GB virus-C and the hepatitis G virus (GBV-C/HGV) are variants of the same positive sense RNA flavivirus, initially thought to be associated with hepatitis. The tissue tropism of GBV-C/HGV in normal subjects has not been evaluated to date using an extended tissue spectrum. Therefore, the sites of GBV-C/HGV replication were investigated in serum and twenty-three tissues collected during post-mortem examination of four apparently healthy individuals who died accidental deaths, who were infected with GBV-C/HGV. All were anti-HIV and anti-HCV negative and three out of four were HBsAg negative. Tissues were collected carefully to prevent cross contamination. A highly strand-specific RT-PCR assay was employed for the detection of either GBV-C/HGV positive strand RNA (virion) or negative strand RNA (replicative intermediary). Strand specificity of the RT-PCR assay was assessed with synthetic positive-and negative strand GBV-C/HGV RNA generated from a plasmid, using T7 and T3 RNA polymerases. The spleen and bone marrow biopsies were found to be uniformly positive for both negative-and positive strand GBV-C/HGV RNA. In addition, one cadaver was positive for both RNA strands in the kidney, and another positive for both in the liver. No negative strand RNA was detected in the following: brain, muscle, heart, thyroid, salivary gland, tonsil, lung, lymph nodes, gall bladder, pancreas, oesophagus, stomach, small bowel, large bowel, adrenal gland, gonad, aorta, skin and cartilage. This preliminary study concludes that GBV-C/HGV is a lymphotropic virus that replicates primarily in the spleen and bone marrow.     

Subcellular distribution of the foot-and-mouth disease virus 3A protein in cells infected with viruses encoding wild-type and bovine-attenuated forms of 3A

Picornavirus infection induces the proliferation and rearrangement of intracellular membranes in response to the synthesis of nonstructural proteins, including 3A. We have previously shown that changes in 3A are associated with the inability of a Taiwanese strain of foot-and-mouth disease virus (FMDV) (OTai) to grow in bovine cells and cause disease in cattle, although the virus grows to high titers in porcine cells and is highly virulent in pigs (C. W. Beard and P. W. Mason, 2000, J. Virol. 74, 987-991). To study if differences in the distribution of 3A could account for the species specificity of OTai, we compared the localization of the OTai 3A with a bovine-virulent 3A (serotype A12) in keratinocytes prepared from the tongues of cattle and pigs. Following either infection of keratinocytes or transfection with 3A we were unable to discern differences in 3A distribution in either species of keratinocyte, independent of the strain of virus (or 3A) utilized. In both cell types, 3A distributed in a pattern that overlapped with an endoplasmic reticulum (ER) marker protein, calreticulin (CRT). Furthermore, although FMDV infection or transfection with 3A did not result in a gross redistribution of CRT, both virus infection and 3A transfection disrupted the Golgi. Other picornaviruses that disrupt Golgi function are sensitive to brefeldin A (BFA), a fungal metabolite that interferes with retrograde transport between the Golgi and the ER. Interestingly, BFA has little effect on FMDV replication, suggesting that FMDV may acquire cellular membranes into its replication complexes in a manner different from that of other picornaviruses.     

Lithium chloride inhibits early stages of foot‐and‐mouth disease virus (FMDV) replication in vitro

Foot‐and‐mouth disease virus (FMDV) causes an economically important and highly contagious disease of cloven‐hoofed animals such as cattle, swine, and sheep. FMD vaccine is the traditional way to protect against the disease, which can greatly reduce its occurrence. However, the use of FMD vaccines to protect early infection is limited. Therefore, the alternative strategy of applying antiviral agents is required to control the spread of FMDV in outbreak situations. As previously reported, LiCl has obviously inhibition effects on a variety of viruses such as transmissible gastroenteritis virus (TGEV), infectious bronchitis coronavirus (IBV), and pseudorabies herpesvirus and EV‐A71 virus. In this study, our findings were the first to demonstrate that LiCl inhibition of the FMDV replication. In this study, BHK‐21 cell was dose‐dependent with LiCl at various stages of FMDV. Virus titration assay was calculated by the 50% tissue culture infected dose (TCID₅₀) with the Reed and Muench method. The cytotoxicity assay of LiCl was performed by the CCK8 kit. The expression level of viral mRNA was measured by RT‐qPCR. The results revealed LiCl can inhibit FMDV replication, but it cannot affect FMDV attachment stage and entry stage in the course of FMDV life cycle. Further studies confirmed that the LiCl affect the replication stage of FMDV, especially the early stages of FMDV replication. So LiCl has potential as an effective anti‐FMDV drug. Therefore, LiCl may be an effective drug for the control of FMDV. Based on that, the mechanism of the antiviral effect of LiCl on FMDV infection is need to in‐depth research in vivo.

     

Murine epidermal Langerhans cells do not express inducible nitric oxide synthase

In Leishmania-infected macrophages (MΦ), the formation of reactive nitrogen intermediates by the inducible isoform of nitric oxide synthase (iNOS) is critical for the killing of the intracellular parasites. We have recently shown that, in addition to MΦ, epidermal Langerhans cells (LC) can phagocytose Leishmania major, but they do not allow parasite replication. Therefore, we analyzed whether LC and MΦ display the same leishmanicidal effector mechanism. Unlike MΦ, stimulation of unselected epidermal cells with interferon-γ/lipopoly-saccharide did not lead to the release of nitric oxide (NO), and inhibition of NO production had no effect on the rate of infection of LC. iNOS mRNA was clearly detectable in MΦ as well as unselected epidermal cells (the majority of which consists of keratinocytes) after stimulation with different cytokines. In contrast, pure LC obtained by single-cell picking from cytokine-activated or L. major-infected epidermal cells did not express iNOS mRNA. Addition of the NO donor S-nitroso-N-acetylpenicillamine to already-infected LC did not alter their rate of infection, indicating that LC do not utilize exogenous NO for the control of intracellular Leishmania. These results suggest that in the L. major-infected skin, activated MΦ and keratinocytes, but not LC have the ability to express iNOS activity. Therefore, an as yet unidentified, NO-independent mechanism appears to be responsible for the control of parasite replication in LC.     

Intracellular membrane proliferation in E. coli induced by foot-and-mouth disease virus 3A gene products

During picornavirus infection replication of genomic RNA occurs in membrane-associated ribonucleoprotein complexes. These replication complexes contain different nonstructural viral proteins with mostly unknown function. To examine the function of nonstructural picornaviral proteins in more detail, cDNA of foot-and-mouth-disease virus (FMDV) strain O1 Lausanne was cloned into lambda ZAP II, and different parts of the P3-coding sequence were expressed in E. coli by the T7 polymerase system. Expression products constituted (a) fusion proteins composed of N-terminal leader peptide of bacteriophage T7 10 protein fused to FMDV P3-sequences of different lengths, (b) translation products of authentic P3-region genes, and (c) carboxy-terminally truncated 3A proteins. Expression products were characterized by NaDodSO₄-polyacrylamide gel electrophoresis, immunoblotting, as well as electron and immunoelectron microscopy. We show here that in the T7 polymerase system a high level of expression of 3A-containing peptides is achieved in E. coli. Remarkably, the expression of 3A-derived proteins induced a dramatic intracellular membrane proliferation in E. coli cells, similar to the vesicle induction observed in FMDV-infected cells. By immunoelectron microscopy, 3A-reactive material was found associated with these membranes. We hypothesize that the FMDV 3A protein is instrumental in eliciting intracellular membrane proliferation in infected cells as a prerequisite for viral RNA replication.     

Expression of class II HLA antigens (HLA-DR, DQW1, DQW3) in normal skin and cutaneous pathology

We report the cutaneous expression of class II HLA antigens disclosed by immunohistochemical staining of normal and lesional skin with monoclonal antibodies (MoAbs) reacting with HLA-DR, DQW1 and DQW3 antigens. Briefly, we disclosed: on normal human skin: 1) The Langerhans cells (LC) HLA-DR+, DQW1+; 2) the acrosyringium HLA-DR+, DQW1+, DQW3+; 3) the dermal vessel endothelial cells HLA-DR+. On lesional skin: 1) The LC were found HLA-DQW3+ in the lesional skin of some cutaneous diseases; this expression was never shown on LC of normal human skin; 2) the epidermal keratinocytes disclosed an uniform membrane expression of HLA-DR antigens in some cutaneous diseases; this kind of expression was not found by immunostaining with MoAbs directed against HLA-DQ antigens; 3) in psoriatic lesions some keratinocytes disclosed an heterogeneous expression of HLA-DR, DQW1 and DQW3 antigens; 4) tumoral cells from cutaneous malignant melanomas were shown to be HLA-DR+, DQW1+, DQW3+. The HLA-DQW3 expression on the LC of lesional skin is in favour with a modulation of HLA-DQW3 expression by unknown factors present in pathological skin. The HLA-DR expression on epidermal keratinocytes suggests a functional collaboration of keratinocytes with LC in the genetic restriction of cutaneous immune reactions.     

Innate immunity mediated by epidermal keratinocytes promotes acquired immunity involving Langerhans cells and T cells in the skin

Skin is an immunological organ consisting of epidermal cells, i.e. keratinocytes and Langerhans cells (LCs, antigen-presenting dendritic cells), and both innate and acquired immune systems operate upon exposure of the skin to various external microbes or their elements. To explore the relationship between innate and acquired immunities in the skin, we investigated whether Toll-like receptor (TLR) ligation of epidermal cells enhances the ability of LCs to present a specific antigen to T cells in mice. LC-containing epidermal cells were incubated with CpG oligonucleotide (TLR9 ligand) modified with trinitrophenyl hapten, and cultured with hapten-primed CD4(+) T cells. TLR9 ligand was capable of enhancing the hapten-presenting ability of LCs when LC-enriched epidermal cells, but not purified LCs, were used as the LC source, suggesting that bystander keratinocytes play a role in the enhancement of LC function. Cultivation of freshly isolated epidermal cells with CpG promoted the expression of major histocompatibility complex (MHC) class II and CD86 molecules on LCs. CpG enhanced the production of interleukin (IL)-1alpha, granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumour necrosis factor (TNF)-alpha by primarily cultured keratinocytes. The addition of a cocktail of neutralizing antibodies against these cytokines abrogated the CpG-promoted, antigen-presenting ability of LC-enriched epidermal cells. Moreover, the addition of culture supernatants from CpG-stimulated keratinocytes restored the ability of purified LCs. Our study demonstrated that although the direct effect of CpG on LCs is minimal, LC function can be up-regulated indirectly by cytokines released by CpG-stimulated keratinocytes. This also implies that innate immunity evoked by TLR ligation of keratinocytes enhances acquired immunity comprising LCs and T cells.     

Immunogenicity and T cell recognition in swine of foot-and-mouth disease virus polymerase 3D

Immunization of domestic pigs with a vaccinia virus (VV) recombinant expressing foot-and-mouth disease virus (FMDV) 3D protein conferred partial protection against challenge with infectious virus. The severity reduction of the clinical symptoms developed by the challenged animals occurred in the absence of significant levels of anti-3D circulating antibodies. This observation suggested that the partial protection observed was mediated by the induction of a 3D-specific cellular immune response. To gain information on the T cell recognition of FMDV 3D protein, we conducted in vitro proliferative assays using lymphocytes from outbred pigs experimentally infected with FMDV and 90 overlapping peptides spanning the complete 3D sequence. The use of pools of two to three peptides allowed the identification of T cell epitopes that were efficiently recognized by lymphocytes from at least four of the five animals analyzed. This recognition was heterotypic because anti-peptide responses increased upon reinfection of animals with a FMDV isolate from a different serotype. The results obtained with individual peptides confirmed the antigenicity observed with peptide pools. Detection of cytokine mRNAs by RT-PCR in lymphocytes stimulated in vitro by individual 3D peptides revealed that IFN-gamma mRNA was the most consistently induced, suggesting that the activated T cells belong to the Th 1 subset. These results indicate that 3D protein contains epitopes that can be efficiently recognized by porcine T lymphocytes from different infected animals, both upon primary and secondary (heterotypic) FMDV infection. These epitopes can extend the repertoire of viral T cell epitopes to be included in subunit and synthetic FMD vaccines.     

A mutant of infectious Asia 1 serotype foot-and-mouth disease virus with the deletion of 10-amino-acid in the 3A protein

Foot-and-mouth disease virus (FMDV) serotype Asia 1 is one of the most predominant endemic serotypes in China. Our previous study has generated a full-length cDNA clone (pBSAs) of an Asia 1 serotype FMDV (As1/CHA/05) isolated from bovine. To further study the properties of this virus, a mutant in the 3A region of the cDNA clone (pBSAs-3A10D), containing the deletion at position 93-102 of the 3A protein of As1/CHA/05, was generated by PCR and cloning. After synthesis of RNA in vitro and transfection, the recombinant rvAs-3A10D virus was recovered from BHK-21 cells. Characterization of the rvAs-3A10D revealed that the infectivity, immunoreactivity, and replication kinetics in BHK-21 and PK-15 cells and virulence in mice of the rvAs-3A10D were similar to that of its parent virus. Notably, while wild-type and recombinant viruses containing full-length sequence of the 3A replicated well in primary calf kidney cells, the mutant rvAs-3A10D failed to replicate in primary calf kidney cells in vitro. Apparently, the full-length sequence of 3A in As1/CHA/05 is a necessary component for its replication in calf kidney cells. The availability of this 3A deletion infectious cDNA clone may help in further investigating the virulent determinants of FMDV and potentially developing FMDV vaccines.