Listeria monocytogenes infection overcomes the requirement for CD40 ligand in exogenous antigen presentation to CD8(+) T cells.

In vivo priming of CD8(+) T lymphocytes against exogenously processed model Ags requires CD4(+) T cell help, specifically interactions between CD40 ligand (CD40L) expressed by activated CD4(+) T cells and CD40, which is present on professional APC such as dendritic cells (DCs). To address this issue in the context of bacterial infection, we examined CD40L-CD40 interactions in CD8(+) T cell priming against an exogenously processed, nonsecreted bacterial Ag. CD40L interactions were blocked by in vivo treatment with anti-CD40L mAb MR-1, which inhibited germinal center formation and CD8(+) T cell cross-priming against an exogenous model Ag, OVA. In contrast, MR-1 treatment did not interfere with CD8(+) T cell priming against a nonsecreted or secreted recombinant Ag expressed by Listeria monocytogenes. Memory and secondary responses of CD8(+) T cells against nonsecreted and secreted bacterial Ags were also largely unimpaired by transient MR-1 treatment. When MR-1-treated mice were concurrently immunized with L. monocytogenes and OVA-loaded splenocytes, cross-priming of OVA-specific naive CD8(+) T cells occurred. No significant decline in cross-priming against OVA was measured when either TNF or IFN-gamma was neutralized in L. monocytogenes-infected animals, demonstrating that multiple signals exist to overcome CD40L blockade of CD8(+) T cell cross-priming during bacterial infection. These data support a model in which DCs can be stimulated in vivo through signals other than CD40, becoming APC that can effectively stimulate CD8(+) T cell responses against exogenous Ags during infection.

Detection and analysis of antigen-specific CD8+ T cells.

Based on recent advances in techniques that can detect and enumerate antigen-specific CD8+ T cells, it is evident that these cells can differentially regulate CD8+ T cell effector mechanisms at the single-cell level. Interplay between effector mechanisms that are employed by antigen-specific CD8+ T cells during the immune response in vivo can be addressed with different techniques that "count" cells either directly (T cell receptor (TCR) expression) or indirectly (antigen-specific cytokine production).

Cutting edge: OFF cycling of TNF production by antigen-specific CD8+ T cells is antigen independent.

Although they are known for their capacity to kill infected cells, Ag-specific CD8(+) T cells elaborate other effector mechanisms, including TNF and IFN-gamma, that contribute to defense against infection. Ag-specific CD8(+) T cells rapidly turn ON and turn OFF IFN-gamma production in direct response to Ag contact, presumably to minimize the potential immunopathology that could result from inappropriate secretion of this inflammatory mediator. In this study, we show, using in vitro propagated and directly ex vivo-analyzed Ag-specific CD8(+) T cells, that in contrast to Ag-dependent ON/OFF cycling of IFN-gamma production, the cessation of TNF production by the same IFN-gamma producing cells is rapid and Ag independent.

Adaptive immunity against Listeria monocytogenes in the absence of type I tumor necrosis factor receptor p55.

Tumor necrosis factor (TNF) and the type I TNF receptor (TNFRI), p55, are critical for resistance against primary infections with the intracellular bacterial pathogen Listeria monocytogenes. Importantly, however, susceptibility to primary listeriosis in cytokine-deficient mice does not preclude the development or expression of effective adaptive immunity against virulent L. monocytogenes. We used TNFRI(-/-) mice to study adaptive antilisterial immunity in the absence of interactions between TNF and TNFRI. Our experiments indicate that TNFRI(-/-) mice survive and clear high-dose challenges with an attenuated strain of L. monocytogenes that is incapable of cell-to-cell spread. Furthermore, TNFRI(-/-) mice immunized with attenuated L. monocytogenes go on to develop potent adaptive immunity to subsequent high-dose challenges with virulent L. monocytogenes. Interestingly, CD8(+) T-cell depletion in vivo inhibits immunity to L. monocytogenes in the spleen but not in the liver of TNFRI(-/-) mice. The adaptive immune response in these animals is characterized by activation of listeriolysin O-specific CD8(+) T cells, which are capable of transferring antilisterial immunity to naive wild-type C57BL/6 host mice. These experiments demonstrate the development and expression of potent CD8(+) T-cell-mediated antilisterial immunity in the absence of TNFRI.

In vitro and in vivo macrophage function can occur independently of SLP-76.

Expression of SH2 domain-containing leukocyte-specific phosphoprotein of 76 kDa (SLP-76), a hematopoietic cell-specific adapter protein, is required to couple Syk family tyrosine kinase activation to downstream mediators such as phospholipase C (PLC)-gamma following TCR, platelet collagen receptor and mast cell Fc epsilon R stimulation. In addition to T cells, mast cells and platelets, SLP-76 is expressed in monocytes and macrophages. To determine the role of SLP-76 in Fc gamma R-stimulated signaling pathways in macrophages, we examined cultured bone marrow-derived macrophages (BMM) from SLP-76(-/-) and wild-type mice. In this study, we show that Fc gamma R cross-linking rapidly induces tyrosine phosphorylation of SLP-76 in wild-type BMM. Surprisingly, however, BMM from SLP-76(-/-) mice activate ERK2 and phosphorylate PLC-gamma 2 following Fc gamma R ligation. Furthermore, SLP-76(-/-) BMM display normal Fc gamma R-dependent phagocytic function and reactive oxygen intermediate production. SLP-76(-/-) and SLP-76(+/+) BMM secrete comparable levels of IL-12 in response to lipopolysaccharide and IFN-gamma. To examine macrophage function in vivo, SLP-76(-/-) mice were challenged i.v. with Listeria monocytogenes. SLP-76(-/-) mice survive and efficiently contain the acute phase of infection similar to wild-type mice but exhibit a stable chronic infection attributed to the lack of mature T cells. These data show that, although SLP-76 is required to couple Syk family PTK activity to downstream mediators and effector functions in Fc gamma R-induced pathways in some cell types, activation of Fc gamma R-dependent pathways occurs independently of SLP-76 in BM

Adaptive immunity and enhanced CD8+ T cell response to Listeria monocytogenes in the absence of perforin and IFN-gamma.

Single Ag-specific CD8+ T cells from IFN-gamma-deficient (GKO) or perforin-deficient (PKO) mice provide substantial immunity against murine infection with Listeria monocytogenes. To address the potential for redundancy between perforin and IFN-gamma as CD8+ T cell effector mechanisms, we generated perforin/IFN-gamma (PKO/GKO) double-deficient mice. PKO/GKO-derived CD8+ T cells specific for the immunodominant listeriolysin O (LLO91-99) epitope provide immunity to LM infection similar to that provided by Ag-matched wild-type (WT) CD8+ T cells in the liver but reduced in the spleen. Strikingly, polyclonal CD8+ T cells from immunized PKO/GKO mice were approximately 100-fold more potent in reducing bacterial numbers than the same number of polyclonal CD8+ T cells from immunized WT mice. This result is probably quantitative, because the frequency of the CD8+ T cell response against the immunodominant LLO91-99 epitope is >4.5-fold higher in PKO/GKO mice than WT mice at 7 days after identical immunizations. Moreover, PKO/GKO mice can be immunized by a single infection with attenuated Listeria to resist >80,000-fold higher challenges with virulent organisms than naive PKO/GKO mice. These data demonstrate that neither perforin nor IFN-gamma is required for the development or expression of adaptive immunity to LM. In addition, the results suggest the potential for perforin and IFN-gamma to regulate the magnitude of the CD8+ T cell response to infection.

Cutting edge: antilisterial activity of CD8+ T cells derived from TNF-deficient and TNF/perforin double-deficient mice.

The mechanisms by which CD8+ T cells mediate immunity against bacterial pathogens remain largely unknown. Perforin-dependent cytolysis plays a role, but is not required for CD8+ T cell-mediated immunity against Listeria monocytogenes. TNF is essential for CD8+ T cell immunity to L. monocytogenes, but the cellular source of TNF is undefined. TNF-deficient and TNF/perforin double-deficient mice were used to generate CD8+ T cells specific for an L. monocytogenes-derived Ag. Wild-type and TNF-deficient CD8+ T cells mediated antilisterial immunity in wild-type but not TNF-deficient host mice, revealing that CD8+ T cell-derived TNF is not required for CD8+ T cell-mediated antilisterial immunity, but demonstrating a role for TNF derived from other cell types. TNF/perforin double-deficient CD8+ T cells mediated antilisterial immunity in the liver, but not in the spleen, of wild-type recipient mice, suggesting that perforin-independent immunity in the spleen requires CD8+ T cell-derived TNF.

Intracellular staining for TNF and IFN-gamma detects different frequencies of antigen-specific CD8(+) T cells.

CD8(+) T lymphocytes are important mediators of adaptive immunity against certain viral, protozoan and bacterial pathogens. Activated CD8(+) T cells are able to induce cytolysis of infected cells (perforin and CD95-CD95L mediated pathways) and also elaborate cytokines, including IFN-gamma and TNF after appropriate MHC class I-peptide recognition. New technologies for the detection of antigen-specific CD8(+) T cells, including tetrameric MHC class I-peptide complexes, intracellular IFN-gamma staining and IFN-gamma ELISPOT analysis have revised our understanding of the magnitude of the CD8(+) T cell response to infection. Here, using intracellular cytokine staining, we compare detection of IFN-gamma and TNF in the analysis of pathogen-specific CD8(+) T cell lines and CD8(+) T cells after primary viral infection (LCMV) or secondary bacterial infection (Listeria monocytogenes). Under multiple conditions and with multiple epitopes, we find that staining for intracellular IFN-gamma consistently detects a higher frequency of antigen-specific CD8(+) T cells than detection of intracellular TNF. However, (a) intracellular staining for TNF can be used to detect antigen-specific CD8(+) T cell responses and (b) intracellular staining for cytokines is a useful approach for in vitro characterization of antigen-specific CD8(+) T cell lines.

CD8(+) T-cell priming against a nonsecreted Listeria monocytogenes antigen is independent of the antimicrobial activities of gamma interferon.

Sublethal infection of mice with recombinant Listeria monocytogenes expressing a model epitope in either secreted or nonsecreted form results in similar CD8(+) T-cell priming. Since nonsecreted bacterial proteins have no obvious access to the endogenous major histocompatibility complex (MHC) class I presentation pathway, presentation of these antigens requires destruction of the bacterium to reveal the nonsecreted molecules to an exogenous MHC class I presentation pathway. Gamma interferon (IFN-gamma), a cytokine made by multiple cell types in response to L. monocytogenes infection, could be required for exogenous presentation of nonsecreted bacterial antigens via its capacity to upregulate the expression of molecules involved in antigen presentation, its capacity to activate macrophages to kill bacteria to expose nonsecreted molecules or both. IFN-gamma knockout (KO) mice were used to address the requirement for IFN-gamma in CD8(+) T-cell priming against (i) a model exogenous antigen and (ii) secreted and nonsecreted L. monocytogenes antigens. We demonstrate that IFN-gamma KO mice are capable of cross-presenting the model exogenous antigen ovalbumin to prime CD8(+) T-cell responses that are only slightly weaker than that in wild-type (WT) mice. Despite their extreme susceptibility to primary L. monocytogenes infection, previously immunized and naive IFN-gamma KO mice were able to generate CD8(+) T-cell responses against both secreted and nonsecreted L. monocytogenes antigens which were similar to responses of WT mice. Interestingly, IFN-gamma KO mice were as capable as WT mice in mediating the characteristic drop in bacterial load in the liver at 4 h postinfection, although the IFN-gamma KO mice have exacerbated bacterial loads as early as 24 h postinfection. These results demonstrate that the regulatory functions of IFN-gamma are not required for priming of CD8(+) T cells by cross-presentation of a model exogenous antigen or in response to a nonsecreted L. monocytogenes antigen. In addition, the capacity of IFN-gamma to activate the microbicidal activities of macrophages is not required for the very early innate immune response to L. monocytogenes or priming of CD8(+) T cells against a nonsecreted bacterial antigen.

Transient expression of bacterial gene fragments in eukaryotic cells: implications for CD8(+) T cell epitope analysis.

CD8(+) T cells are potent effectors of acquired immunity against some viruses and intracellular bacterial pathogens. Antigens recognized by CD8(+) T cells are small, 8-9 amino acid peptides derived from proteins produced by the pathogen. These peptides are presented by MHC class I molecules on the surface of the infected cell. When characterizing the CD8(+) T cell response to a bacterial or viral pathogen, it is often necessary to express an antigenic protein in a eukaryotic host cell that is capable of processing and presenting peptide epitopes to antigen-specific CD8(+) T cells. We describe a system designed to transiently express bacterial polypeptides and MHC class I molecules in eukaryotic cells. Recognition of these peptide-MHC complexes stimulates TNF production by antigen-specific CD8(+) T cell lines. This system should be useful for analysis of CD8(+) T cell epitope-containing bacterial gene fragments when expression of the entire bacterial protein is detrimental to the eukaryotic cell, or when overexpression of the bacterial gene is detrimental to the bacterial cloning strain. Furthermore, this system can be used for the rapid mapping of CD8(+) T cell epitopes within a protein.

Perforin-deficient CD8+ T cells: in vivo priming and antigen-specific immunity against Listeria monocytogenes.

CD8+ T cells require perforin to mediate immunity against some, but not all, intracellular pathogens. Previous studies with H-2b MHC perforin gene knockout (PO) mice revealed both perforin-dependent and perforin-independent pathways of CD8+ T cell-mediated immunity to Listeria monocytogenes (LM). In this study, we address two previously unresolved issues regarding the requirement for perforin in antilisterial immunity: 1) Is CD8+ T cell-mediated, perforin-independent immunity specific for a single Ag or generalizable to multiple Ags? 2) Is there a deficiency in the priming of the CD8+ T cell compartment of PO mice following an immunizing challenge with LM? We used H-2d MHC PO mice to generate CD8+ T cell lines individually specific for three known Ags expressed by a recombinant strain of virulent LM. Adoptive transfer experiments into BALB/c host mice revealed that immunity can be mediated by PO CD8+ T cells specific for all Ags examined, indicating that perforin-independent immunity is not limited to CD8+ T cells that recognize listeriolysin O. Analysis of epitope-specific CD8+ T cell expansion by MHC class I tetramer staining and ELISPOT revealed no deficiency in either the primary or secondary response to LM infection in PO mice. These results demonstrate that the perforin-independent pathway of antilisterial resistance mediated by CD8+ T cells is generalizable to multiple epitopes. Furthermore, the results show that reduced antilisterial resistance observed with polyclonal PO CD8+ T cells is a consequence of a deficiency in effector function and not a result of suboptimal CD8+ T cell priming.

A knockout approach to understanding CD8+ cell effector mechanisms in adaptive immunity to Listeria monocytogenes.

In the described experimental approach, we use an attenuated LM strain to evoke LM specific CD8+ T cell responses. In this fashion, we can immunize immunocompromised gene knockout mice, that would succumb to low level infection with virulent LM. We then generate antigen matched, LM-specific CD8+ T cell lines from wild-type and gene knockout mice, and compare their capacity to provide immunity to LM infection in vivo. To date, our results demonstrate that CD8+ T cell-derived IFN-gamma and TNF are not required effector functions. Perforin deficiency has an impact on CD8+ T cell immunity but our studies provide strong evidence for the existence of perforin independent pathways of CD8+ T cell immunity to LM. To assess the potential for redundancy in effector mechanisms, we have generated mice deficient in both perforin and IFN-gamma and are developing mice deficient in perforin and TNF. By removing the major CD8+ T cell effector mechanisms, singly and in combination, we will eventually determine whether immunity to LM can be provided by redundant effector pathways or if novel effector mechanisms exist beyond our current knowledge. The generation of MHC matched, single and double knockout mice, will also aid in continuing studies to analyze the role of these molecules in resistance to in vivo infection.

Interactions of the invasive pathogens Salmonella typhimurium, Listeria monocytogenes, and Shigella flexneri with M cells and murine Peyer's patches.

Invasive enteric bacteria must pass through the intestinal epithelium in order to establish infection. It is becoming clear that a common target for intestinal mucosa penetration is the specialized epithelial cell of Peyer's patches, the M cell. In order to gain a better understanding of how bacteria interact with M cells, we have compared the interactions of Salmonella typhimurium, Listeria monocytogenes, and Shigella flexneri with M cells by using a murine ligated-loop model. Our results indicate that S. typhimurium possesses a highly efficient mechanism for M cell entry that targets and destroys these cells, while L. monocytogenes and S. flexneri appear to be internalized into M cells in a less disruptive fashion. Early uptake of Listeria or Shigella into M cells appeared to lead to the death of some cells, as evidenced by the appearance of holes in the intestinal epithelium. At later time points, the follicle-associated epithelium of animals infected with these bacteria displayed extensive destruction. These data indicate that enteric pathogens use different strategies to interact with M cells and initiate infection of a host.

Perforin-deficient CD8+ T cells provide immunity to Listeria monocytogenes by a mechanism that is independent of CD95 and IFN-gamma but requires TNF-alpha.

CD8+ T cells are effective mediators of immunity against Listeria monocytogenes, but the mechanisms by which they provide antilisterial immunity are poorly understood. CD8+ T cells efficiently lyse target cells in vitro by at least two independent pathways. To test the hypothesis that CD8+ T cell-mediated immunity to L. monocytogenes is dependent on perforin or CD95 (Fas, Apo-1), we used C57BI/6 (B6) and perforin-deficient (PO) mice to generate CD8+ T cell lines specific for the L. mono cytogenes-encoded Ag listeriolysin O (LLO). Both lines specifically produce IFN-gamma and TNF-alpha, and mediate target cell lysis in vitro. Cytolysis mediated by the PO-derived CD8+ T cell line is delayed relative to the B6-derived line and is completely inhibited by anti-CD95 Abs. In vivo, PO-derived CD8+ T cells provide specific antilisterial immunity in B6 hosts, CD95-deficient hosts, and IFN-gamma-depleted hosts. However, PO-derived CD8+ T cells fail to provide antilisterial immunity in hosts depleted of TNF-alpha. These results indicate that single Ag-specific CD8+ T cells derived from PO mice can mediate antilisterial immunity by a mechanism that is independent of CD95 or IFN-gamma, but requires TNF-alpha.

CD8 T-cell recognition of macrophages and hepatocytes results in immunity to Listeria monocytogenes.

CD8 T cells are effective mediators of specific immunity to infection by Listeria monocytogenes, a bacterial pathogen that initially infects macrophages in the spleen and liver and subsequently spreads to hepatocytes and unidentified parenchymal cells in the spleen. To identify the in vivo target cells of L. monocytogenes-immune CD8 T cells, adoptive transfer assays were performed with bone marrow chimeric or transgenic host mice which had been manipulated to alter the major histocompatibility complex molecules expressed on macrophages or hepatocytes. L. monocytogenes-immune CD8 T cells mediate significant immunity in BDF1-->beta 2 M-/- chimeras, comparable to that seen in unmanipulated BDF1 recipients. L. monocytogenes-immune CD8 T cells also mediate significant antilisterial immunity in parent-->F1 chimeras when the CD8 T cells are syngeneic with the bone marrow donor. These data demonstrate that bone marrow-derived macrophages are major targets for L. monocytogenes-immune CD8 T cells in adoptive transfer assays. Interestingly, significant immunity was observed in parent-->F1 chimeras when the L. monocytogenes-immune CD8 T cells were not syngeneic with the bone marrow donor, suggesting that recognition of Listeria-infected non-bone-marrow-derived cells such as hepatocytes may also occur in vivo. Consistent with this possibility, H-2Kb-restricted CD8 T cells specific for the listeriolysin O molecule mediate significant immunity in the liver, but not the spleen, in transgenic mice expressing H-2Kb only on hepatocytes. In addition, Listeria-specific CD8 T cells lyse Listeria-infected hepatocyte-like cells in vitro. Thus, Listeria-infected hepatocytes can be recognized by CD8 T cells in vivo and in vitro.

C58 and AKR mice of all ages develop motor neuron disease after lactate dehydrogenase-elevating virus infection but only if antiviral immune responses are blocked by chemical or genetic means or as a result of old age.

Age-dependent poliomyelitis is a paralytic disease of C58 and AKR mice caused by cytocidal infection of anterior horn neurons with neuropathogenic strains of lactate dehydrogenase-elevating virus (LDV). The motor neurons are rendered LDV-permissive via an unknown mechanism through the expression of ecotropic murine leukemia virus (MuLV) in central nervous system (CNS) glial cells. Only old mice develop paralytic disease after LDV infection, but mice 5-6 months old or older can be rendered susceptible by suppression of anti-LDV immune responses by a single treatment with cyclophosphamide or X-irradiation before LDV infection. Younger mice appeared to be resistant in spite of this immunosuppresive treatment. The present results confirm that mice as young as 1 month of age possess CNS cells expressing ecotropic MuLV and show that these mice are susceptible to paralytic LDV infection provided their anti-LDV immune responses are blocked for an extended period of time by repeated cyclophosphamide treatments or by a genetic defect. Furthermore, old mice become naturally susceptible to paralytic LDV infection because of an impaired ability to mount a motor neuron protective anti-LDV immune response.

A positively selecting thymic epithelial cell line lacks costimulatory activity.

The participation of costimulatory molecule interactions in positive selection of T lymphocytes was addressed by assessing the ability of a positively selecting thymic epithelial cell (TEC) line, 427.1, to stimulate allospecific CTL responses. Stimulation of H-2s spleen cells with the H-2b expressing 427.1 line does not result in the generation of cells capable of lysing H-2b target cell lines. The level of expression of MHC class I molecules by 427.1 is lower than that found in other stimulatory TEC lines. However, this finding does not account for the nonstimulatory phenotype. Up-regulation of MHC class I did not result in stimulation, and fusion of 427.1 cells with stimulatory TEC resulted in a line with low MHC class I molecule expression and stimulatory phenotype. The TEC line 427.1 does not express the costimulatory molecule B7/BB1, and transfection of the B7/BB1-encoding DNA results in expression of the molecule and conversion into a stimulatory phenotype, demonstrating directly that the non-stimulatory phenotype is a result of lack of costimulation. However, B7/BB1 expression does not improve the ability of 427.1 TECs to induce positive selection. Intrathymic injection of the B7/BB1 transfected, compared with mock transfected 427.1 cells, rescued fewer CD8+ mature thymocytes in beta 2-microglobulin negative mice. Therefore, unlike the peripheral T cell responses to Ag, positive selection of T cells in the thymus may not depend on the costimulation provided by the presenting cell.

CD8 T cells can protect against an intracellular bacterium in an interferon gamma-independent fashion.

Specific T-cell immunity to Listeria monocytogenes is thought to occur through the action of lymphokines which activate phagocytes to ingest and kill microorganisms. Interferon gamma (IFN-gamma) has been shown to be an effective mediator of this type of macrophage activation in vivo and in vitro. The monoclonal antibody H22.1 efficiently neutralizes endogenous IFN-gamma, exacerbates disease in a mouse model of L. monocytogenes infection, and inhibits the in vivo protective activity of a Listeria antigen-specific CD4 T-cell line. In contrast, in vivo protection by Listeria-immune CD8 T cells is not inhibited by the neutralizing anti-IFN-gamma monoclonal antibody. These results suggest that CD8 T cells can protect against an intracellular pathogen in an IFN-gamma-independent manner.

Precise prediction of a dominant class I MHC-restricted epitope of Listeria monocytogenes.

Listeria monocytogenes is a Gram-positive bacterium which grows in the cytoplasm of eukaryotic cells and can cause severe disease in immunocompromised individuals. In murine systems CD8+ T lymphocytes have been shown to be important effectors of acquired protective immunity against L. monocytogenes. Class I MHC-restricted CD8+ cytotoxic T lymphocytes (CTL), which lyse J774 macrophage-like targets infected with L. monocytogenes, are induced following in vivo injection of live organisms. Natural peptide epitopes derived from L. monocytogenes can be acid-extracted from heavily infected BALB/c spleens and detected by CTL. A CTL clone, B9, derived from a (BALB/c x C57BL/6)F1 (H-2dxb) mouse, recognizes one of these natural epitopes in an H-2Kd-restricted fashion. B9 also recognizes P815 (H-2d) mastocytoma cells transfected with the listeriolysin gene. To identify the region of the listeriolysin recognized by CTL we used the H-2Kd peptide-binding motif described by Rammensee and colleagues to synthesize 11 nonamer peptides. One of these peptides, listeriolysin 91-99, was recognized very efficiently by B9. This represents the first identified class I MHC-restricted epitope of bacteria and demonstrates the utility of the allele-specific motif for predicting CTL epitopes.

A nested set of eight RNAs is formed in macrophages infected with lactate dehydrogenase-elevating virus.

Total RNA was extracted from primary cultures of mouse macrophages isolated from 10-day-old mice 6 to 12 h postinfection with lactate dehydrogenase-elevating virus (LDV). Poly(A)+ RNA was extracted from spleens of 18-h LDV-infected mice. The RNAs were analyzed by Northern (RNA) blot hybridization with a number of LDV-specific cDNAs as probes. A cDNA representing the nucleocapsid protein (VP-1) gene located at the 3' terminus of the viral genome (E. K. Godeny, D. W. Speicher, and M. A. Brinton, Virology 177:768-771, 1990) hybridized to viral genomic RNA of about 13 kb plus seven subgenomic RNAs ranging in size from about 1 to about 3.6 kb. Two other cDNA clones hybridized only to the four or five largest subgenomic RNAs, respectively. In contrast, two cDNAs encoding continuous open reading frames with replicase and zinc finger motifs hybridized only to the genomic RNA. The replicase motif exhibited 75% amino acid identity to that of the 1b protein of equine arteritis virus (EAV) and 44% amino acid identity to those of the 1b proteins of coronaviruses and Berne virus. Combined, the results indicate that LDV replication involves formation of a 3'-coterminal-nested set of mRNAs as observed for coronaviruses and toroviruses as well as for EAV, with which LDV shares many other properties. Overall, LDV, like EAV, possesses a genome organization resembling that of the coronaviruses and toroviruses. However, EAV and LDV differ from the latter in the size of their genomes, virion size and structure, nature of the structural proteins, and symmetry of the nucleocapsids.