Neisseria gonorrhoeae PilV, a type IV pilus-associated protein essential to human epithelial cell adherence.

Type IV pili (Tfp) of Neisseria gonorrhoeae, the Gram-negative etiologic agent of gonorrhea, facilitate colonization of the human host. Tfp are assumed to play a key role in the initial adherence to human epithelial cells by virtue of the associated adhesin protein PilC. To examine the structural and functional basis for adherence in more detail, we identified potential genes encoding polypeptides sharing structural similarities to PilE (the Tfp subunit) within the N. gonorrhoeae genome sequence database. We show here that a fiber subunit-like protein, termed PilV, is essential to organelle-associated adherence but dispensable for Tfp biogenesis and other pilus-related phenotypes, including autoagglutination, competence for natural transformation, and twitching motility. The adherence defect in pilV mutants cannot be attributed to reduced levels of piliation, defects in fiber anchoring to the bacterial cell surface, or to unstable pilus expression related to organelle retraction. PilV is expressed at low levels relative to PilE and copurifies with Tfp fibers in a PilC-dependent fashion. Purified Tfp from pilV mutants contain PilC adhesin at reduced levels. Taken together, these data support a model in which PilV functions in adherence by promoting the functional display of PilC in the context of the pilus fiber.

Conformational change, aggregation and fibril formation induced by detergent treatments of cellular prion protein.

The conversion of protease-sensitive prion protein (PrP-sen) to a high beta-sheet, protease-resistant and often fibrillar form (PrP-res) is a central event in transmissible spongiform encephalopathies (TSE) or prion diseases. This conversion can be induced by PrP-res itself in cell-free conversion reactions. The detergent sodium N-lauroyl sarkosinate (sarkosyl) is a detergent that is widely used in PrP-res purifications and is known to stimulate the PrP-res-induced conversion reaction. Here we report effects of sarkosyl and other detergents on recombinant hamster PrP-sen purified from mammalian cells under oxidizing conditions that maintain the single native disulfide bond. Low concentrations of sarkosyl (0.001-0.1%) induced aggregation of PrP-sen molecules, increased light scattering, altered fluorescence excitation and emission spectra, and enhanced the proportion of beta-sheet secondary structure according to circular dichroism and infrared spectroscopies. An enhancement of beta-sheet content was also seen with 0.001% sodium dodecyl sulfate (SDS) but not several other types of detergents. Electron microscopy revealed that sarkosyl induced the formation of both amorphous and fibrillar aggregates. The fibrils appeared to be constructed from spherical bead-like protofibrils. Neither TSE infectivity nor the characteristic partial proteinase K resistance of PrP-res was detected in the sarkosyl-induced PrP aggregates. We conclude that certain anionic detergents can disrupt the conformation of PrP-sen and induce high beta-sheet aggregates that are distinct from scrapie-associated PrP-res in terms of protease-resistance, infrared spectrum and infectivity. These results reinforce the idea that not all high-beta aggregates of PrP are equivalent to the pathologic form, PrP-res.

Structural alterations in a type IV pilus subunit protein result in concurrent defects in multicellular behaviour and adherence to host tissue.

The ability of bacteria to establish complex communities on surfaces is believed to require both bacterial-substratum and bacterial-bacterial interactions, and type IV pili appear to play a critical but incompletely defined role in both these processes. Using the human pathogen Neisseria gonorrhoeae, spontaneous mutants defective in bacterial self-aggregative behaviour but quantitatively unaltered in pilus fibre expression were isolated by a unique selective scheme. The mutants, carrying single amino acid substitutions within the conserved amino-terminal domain of the pilus fibre subunit, were reduced in the ability to adhere to a human epithelial cell line. Co-expression of the altered alleles in the context of a wild-type pilE gene confirmed that they were dominant negative with respect to aggregation and human cell adherence. Strains expressing two copies of the altered alleles produced twice as much purifiable pili but retained the aggregative and adherence defects. Finally, the defects in aggregative behaviour and adherence of each of the mutants were suppressed by a loss-of-function mutation in the twitching motility gene pilT. The correlations between self-aggregation and the net capacity of the microbial population to adhere efficiently demonstrates the potential significance of bacterial cell-cell interactions to colonization.

Identification of aleutian mink disease parvovirus capsid sequences mediating antibody-dependent enhancement of infection, virus neutralization, and immune complex formation.

Aleutian mink disease parvovirus (ADV) causes a persistent infection associated with circulating immune complexes, immune complex disease, hypergammaglobulinemia, and high levels of antiviral antibody. Although antibody can neutralize ADV infectivity in Crandell feline kidney cells in vitro, virus is not cleared in vivo, and capsid-based vaccines have proven uniformly ineffective. Antiviral antibody also enables ADV to infect macrophages, the target cells for persistent infection, by Fc-receptor-mediated antibody-dependent enhancement (ADE). The antibodies involved in these unique aspects of ADV pathogenesis may have specific targets on the ADV capsid. Prominent differences exist between the structure of ADV and other, more-typical parvoviruses, which can be accounted for by short peptide sequences in the flexible loop regions of the capsid proteins. In order to determine whether these short sequences are targets for antibodies involved in ADV pathogenesis, we studied heterologous antibodies against several peptides present in the major capsid protein, VP2. Of these antibodies, a polyclonal rabbit antibody to peptide VP2:428-446 was the most interesting. The anti-VP2:428-446 antibody aggregated virus particles into immune complexes, mediated ADE, and neutralized virus infectivity in vitro. Thus, antibody against this short peptide can be implicated in key facets of ADV pathogenesis. Structural modeling suggested that surface-exposed residues of VP2:428-446 are readily accessible for antibody binding. The observation that antibodies against a single target peptide in the ADV capsid can mediate both neutralization and ADE may explain the failure of capsid-based vaccines.

N-terminal cleavage fragment of glycosylated Gag is incorporated into murine oncornavirus particles.

Glycosylated Gag (Glycogag) is a transmembrane protein encoded by murine and feline oncornaviruses. While the protein is dispensible for virus replication, Glycogag-null mutants of a neurovirulent murine oncornavirus are slow to spread in vivo and exhibit a loss of pathogenicity. The function of this protein in the virus life cycle, however, is not understood. Glycogag is expressed at the plasma membrane of infected cells but has not been detected in virions. In the present study we have reexamined this issue and have found an N-terminal cleavage fragment of Glycogag which was pelleted by high-speed centrifugation and sedimented in sucrose density gradients at the same bouyant density as virus particles. Its association with virions was confirmed by velocity sedimentation through iodixanol, which effectively separated membrane microvesicles from virus particles. Furthermore, the apparent molecular weight of the virion-associated protein was different from that of the protein extracted from the plasma membrane, suggesting some level of specificity or selectivity of incorporation.

Developmental expression of two spore wall proteins during maturation of the microsporidian Encephalitozoon intestinalis.

Microsporidia are intracellular eukaryotes that infect many animals and cause opportunistic infections in AIDS patients. The disease is transmitted via environmentally resistant spores. Two spore wall constituents from the microsporidian Encephalitozoon intestinalis were characterized. Spore wall protein 1 (SWP1), a 50-kDa glycoprotein recognized by monoclonal antibody (MAb) 11B2, was detected in developing sporonts and at low levels on the surfaces of mature spores. In contrast, SWP2, a 150-kDa glycoprotein recognized by MAb 7G7, was detected on fully formed sporonts and was more abundant on mature spores than SWP1. Nevertheless, the SWPs appeared to be complexed on the surfaces of mature spores. SWP1 and SWP2 are similar at the DNA and protein levels and have 10 conserved cysteines in the N-terminal domain, suggesting similar secondary structures. The C-terminal domain of SWP2 has a unique region containing 50 repeating 12- or 15-amino-acid units that lacks homology to known protein motifs. Antibodies from mice infected with E. intestinalis recognized SWP1 and SWP2. The characterization of two immunogenic SWPs from E. intestinalis will allow the study of exospore structure and function and may lead to the development of useful tools in the diagnosis and treatment of microsporidiosis.

Modulation of gonococcal piliation by regulatable transcription of pilE.

The gonococcal pilus, a member of the type IV family of pili, is composed of numerous monomers of the pilin protein and plays an important role in the initiation of disease by providing the primary attachment of the bacterial cell to human mucosal tissues. Piliation also correlates with efficient DNA transformation. To investigate the relationships between these pilus-related functions, the piliation state, and the availability of pilin, we constructed a derivative of MS11-C9 (DeltapilE1) in which the lacIOP regulatory sequences control pilE transcription. In this strain, MS11-C9.10, the steady-state levels of pilin mRNA and protein directly correlate with the concentration of IPTG (isopropyl-beta-D-thiogalactopyranoside) in the growth medium and can reach near-wild-type levels of expression. Transmission electron microscopy (TEM) demonstrated that the number of pili per cell correlated with the steady-state expression levels: at a low level of transcription, single long pili were observed; at a moderate expression level, many singular and bundled pili were expressed; and upon full gene expression, increased lateral association between pili was observed. Analysis of pilus assembly by TEM and epithelial cell adherence over a time course of induction demonstrated that pili were expressed as early as 1 h postinduction. Analysis at different steady-state levels of transcription demonstrated that DNA transformation efficiency and adherence of MS11-C9.10 to transformed and primary epithelial cells also correlated with the level of piliation. These data show that modulation of the level of pilE transcription, without a change in pilE sequence, can alter the number of pili expressed per cell, pilus bundling, DNA transformation competence, and epithelial cell adherence of the gonococcus.

Components and dynamics of fiber formation define a ubiquitous biogenesis pathway for bacterial pili.

Type IV pili (Tfp) are a unique class of multifunctional surface organelles in Gram-negative bacteria, which play important roles in prokaryotic cell biology. Although components of the Tfp biogenesis machinery have been characterized, it is not clear how they function or interact. Using Neisseria gonorrhoeae as a model system, we report here that organelle biogenesis can be resolved into two discrete steps: fiber formation and translocation of the fiber to the cell surface. This conclusion is based on the capturing of an intermediate state in which the organelle is retained within the cell owing to the simultaneous absence of the secretin family member and biogenesis component PilQ and the twitching motility/pilus retraction protein PilT. This finding is the first demonstration of a specific translocation defect associated with loss of secretin function, and additionally confirms the role of PilT as a conditional antagonist of stable pilus fiber formation. These findings have important implications for Tfp structure and function and are pertinent to other membrane translocation systems that utilize a highly related set of components.

Bacteriophages of spirochetes.

Historically, a number of bacteriophage-like particles have been observed in association with members of the bacterial order Spirochetales, the spirochetes. In the last decade, several spirochete bacteriophages have been isolated and characterized at the molecular level. We have recently characterized a bacteriophage of the Lyme disease agent, Borrelia burgdorferi, which we have designated phiBB-1. Here we review the history of the association between the spirochetes and their bacteriophages, with a particular emphasis on phiBB-1 and its prophage, the 32-kb circular plasmid family of B. burgdorferi.

Ultrastructure of Rickettsia rickettsii actin tails and localization of cytoskeletal proteins.

Actin-based motility (ABM) is a mechanism for intercellular spread that is utilized by vaccinia virus and the invasive bacteria within the genera Rickettsia, Listeria, and Shigella. Within the Rickettsia, ABM is confined to members of the spotted fever group (SFG), such as Rickettsia rickettsii, the agent of Rocky Mountain spotted fever. Infection by each agent induces the polymerization of host cell actin to form the typical F (filamentous)-actin comet tail. Assembly of the actin tail propels the pathogen through the host cytosol and into cell membrane protrusions that can be engulfed by neighboring cells, initiating a new infectious cycle. Little is known about the structure and morphogenesis of the Rickettsia rickettsii actin tail relative to Shigella and Listeria actin tails. In this study we examined the ultrastructure of the rickettsial actin tail by confocal, scanning electron, and transmission electron microscopy. Confocal microscopy of rhodamine phalloidin-stained infected Vero cells revealed the typhus group rickettsiae, Rickettsia prowazekii and Rickettsia typhi, to have no actin tails and short (approximately 1- to 3-micrometer) straight or hooked actin tails, respectively. The SFG rickettsia, R. rickettsii, displayed long actin tails (>10 micrometer) that were frequently comprised of multiple, distinct actin bundles, wrapping around each other in a helical fashion. Transmission electron microscopy, in conjunction with myosin S1 subfragment decoration, revealed that the individual actin filaments of R. rickettsii tails are >1 micrometer long, arranged roughly parallel to one another, and oriented with the fast-growing barbed end towards the rickettsial pole. Scanning electron microscopy of intracellular rickettsiae demonstrated R. rickettsii to have polar associations of cytoskeletal material and R. prowazekii to be devoid of cytoskeletal interactions. By indirect immunofluorescence, both R. rickettsii and Listeria monocytogenes actin tails were shown to contain the cytoskeletal proteins vasodilator-stimulated phosphoprotein profilin, vinculin, and filamin. However, rickettsial tails lacked ezrin, paxillin, and tropomyosin, proteins that were associated with actin tails of cytosolic or protrusion-bound Listeria. The unique ultrastructural and compositional characteristics of the R. rickettsii actin tail suggest that rickettsial ABM is mechanistically different from previously described microbial ABM systems.

Invasion and intracellular development of the human granulocytic ehrlichiosis agent in tick cell culture.

Human granulocytotropic ehrlichias are tick-borne bacterial pathogens that cause an acute, life-threatening illness, human granulocytic ehrlichiosis (HGE). Ehrlichias within neutrophil granulocytes that invade tick bite sites are likely ingested by the vector, to be transmitted to another mammalian host during the tick's next blood meal. Thus, the cycle of replication and development in the vector is prerequisite to mammalian infection, and yet these events have not been described. We report tick cell culture isolation of two strains of the HGE agent directly from an infected horse and a dog and have also established a human isolate from HL60 culture in tick cells, proving that the blood stages of the HGE agent are infectious for tick cells, as are those replicating in the human cell line HL60. This required changes to the culture system, including a new tick cell line. In tick cell layers, the HGE agent induced foci of infection that caused necrotic plaques and eventual destruction of the culture. Using the human isolate and electron microscopy, we monitored adhesion, internalization, and replication in vector tick cells. Both electron-lucent and -dense forms adhered to and entered cells by a mechanism reminiscent of phagocytosis. Ehrlichial cell division was initiated soon after, resulting in endosomes filled with numerous ehrlichias. During early development, pale ehrlichias with a tight cell wall dominated, but by day 2, individual bacteria condensed into dark forms with a rippled membrane. These may become compacted into clumps where individual organisms are barely discernible. Whether these are part of an ehrlichia life cycle or are degenerating is unknown.

Estrogen induces cytokeratin aggregation in primary cultures of Armenian hamster hepatocytes.

The effect of estrogen administration to cultured Armenian hamster was studied. Isolated Armenian hamster hepatocytes were cultured in RPMI medium supplemented with beta-estradiol (E2). Beta-estradiol treatment for 24-48 hr induced cytoplasmic inclusion bodies which by immunocytochemistry were positive for cytokeratin (CK) 8, CK 18, and ubiquitin but negative for CK 7 and CK 19. These inclusion bodies appeared as filamentous tangles or amorphous aggregates when observed by electron microscopy. F-actin, tubulin, and desmosomes were not influenced by the presence of the inclusion bodies. Addition of ethanol to culture medium increased the incidence of the inclusion formation. In combination with 0.5% ethanol 1 microM of E2 induced five to six times more inclusion bodies, while the number of inclusion bodies decreased when epidermal growth factor (EGF) was added to the medium in combination with E2. This reduction effect was nullified by treatment with anti-EGF receptor antibody. These findings suggest that E2 treatment to Armenian hamster hepatocytes in vitro induces Mallory body-like inclusions whose incidence can be influenced by addition of ethanol or EGF to the culture medium.

The comP locus of Neisseria gonorrhoeae encodes a type IV prepilin that is dispensable for pilus biogenesis but essential for natural transformation.

The expression of type IV pili (Tfp) by Neisseria gonorrhoeae has been shown to be essential for natural genetic transformation at the level of sequence-specific uptake of DNA. All previously characterized mutants defective in this step of transformation either lack Tfp or are altered in the expression of Tfp-associated properties, such as twitching motility, autoagglutination and the ability to bind to human epithelial cells. To examine the basis for this relationship, we identified potential genes encoding polypeptides sharing structural similarities to PilE, the Tfp subunit, within the N. gonorrhoeae genome sequence database. We found that disruption of one such gene, designated comP (for competence-associated prepilin), leads to a severe defect in the capacity to take up DNA in a sequence-specific manner, but does not alter Tfp biogenesis or expression of the Tfp-associated properties of auto-agglutination, twitching motility and human epithelial cell adherence. Indirect evidence based on immunodetection suggests that ComP is expressed at very low levels relative to that of PilE. The process of DNA uptake in gonococci, therefore, is now known to require the expression of at least three distinct components: Tfp, the recently identified PilT protein and ComP.

Leukocyte infection by the granulocytic ehrlichiosis agent is linked to expression of a selectin ligand.

Human granulocytic ehrlichiosis (HGE) is an emerging tickborne illness caused by an intracellular bacterium that infects neutrophils. Cells susceptible to HGE express sialylated Lewis x (CD15s), a ligand for cell selectins. We demonstrate that adhesion of HGE to both HL60 cells and normal bone marrow cells directly correlates with their CD15s expression. HGE infection of HL60 cells, bone marrow progenitors, granulocytes, and monocytes was blocked by monoclonal antibodies against CD15s. However, these antibodies did not inhibit HGE binding, and anti-CD15s was capable of inhibiting the growth of HGE after its entry into the target cell. In contrast, neuraminidase treatment of HL60 cells prevented both HGE binding and infection. A cloned cell line (HL60-A2), derived from HL60 cells and resistant to HGE, was deficient in the expression of alpha-(1, 3)fucosyltransferase (Fuc-TVII), an enzyme known to be required for CD15s biosynthesis. Less than 1% of HL60-A2 cells expressed CD15s, and only these rare CD15s-expressing cells bound HGE and became infected. After transfection with Fuc-TVII, cells regained CD15s expression, as well as their ability to bind HGE and become infected. Thus, CD15s expression is highly correlated with susceptibility to HGE, and it, and/or a closely related sialylated and alpha-(1,3) fucosylated molecule, plays a key role in HGE infection, an observation that may help explain the organism's tropism for leukocytes.

Suppression of an absolute defect in type IV pilus biogenesis by loss-of-function mutations in pilT, a twitching motility gene in Neisseria gonorrhoeae.

Type IV pili of Neisseria gonorrhoeae, the Gram-negative etiologic agent of gonorrhea, facilitate colonization of the human host. Gonococcal PilT, a protein belonging to a large family of molecules sharing a highly conserved nucleotide binding domain motif, has been shown to be dispensable for organelle biogenesis but essential for twitching motility and competence for genetic transformation. Here, we show that the defect in pilus biogenesis resulting from mutations in the pilC gene, encoding a putative pilus-associated adhesin for human tissue, can be suppressed by the absence of functional PilT. These data conclusively demonstrate that PilT influences the Type IV pilus biogenesis pathway and strongly suggest that organelle expression is a dynamic process. In addition, these findings imply that PilT antagonizes the process of organelle biogenesis and provide the basis for a model for how the counteractive roles of PilT and PilC might relate mechanistically to the phenomenon of twitching motility.

Monocytic differentiation inhibits infection and granulocytic differentiation potentiates infection by the agent of human granulocytic ehrlichiosis.

Human granulocytic ehrlichiosis (HGE) is an emerging tick-borne infection with a specific tropism for granulocytes. We previously isolated and cultivated the HGE agent in the promyelocytic leukemia cell line HL-60 and have also demonstrated the susceptibility of both granulocytic and monocytic human marrow progenitors. Circulating monocytes have not been observed to be infected, suggesting that cell susceptibility may be differentiation specific. To evaluate this hypothesis, HL-60 cells were differentiated towards granulocytes (with dimethyl sulfoxide or all-trans retinoic acid) or toward monocytes-macrophages (with 12-O-tetradecanoylphorbol-13-acetate [TPA], gamma interferon, or 1, 25-dihydroxyvitamin D3) and then challenged with HGE. HGE binding, internalization, and proliferation were compared in differentiated and untreated control HL-60 cells by immunofluorescence, electron microscopy, and Giemsa staining. Granulocytic differentiation resulted in a doubling of HGE binding and enhanced infection consistent with the agent's clinical tropism for neutrophils. Granulocytic cells were unable to kill internalized ehrlichiae even after activation induced by N-formyl-Met-Leu-Phe alone or together with tumor necrosis factor alpha. In contrast, monocyte-macrophage differentiation with TPA resulted in complete resistance to infection through at least two distinct mechanisms: (i) reduction in binding and uptake and (ii) killing of any internalized organisms. Diminished binding in TPA-treated cells correlated with their reduced expression of sialyl Lewis x (CD15s), a putative cellular receptor component for HGE. The degree of monocytic differentiation and activation induced (i.e., TPA > gamma interferon > vitamin D3) correlated with resistance to HGE. Thus, HL-60 cells exhibit a striking differentiation-specific susceptibility to HGE. Differentiation-induced changes in bacterial adhesion and killing capacity underlie the tropism of HGE for granulocytic HL-60 cells and, conversely, the resistance of activated macrophages to infection.

Rhodamine-123 staining in hematopoietic stem cells of young mice indicates mitochondrial activation rather than dye efflux.

Low-intensity fluorescence of rhodamine-123 (Rh-123) discriminates a quiescent hematopoietic stem cell (HSC) population in mouse bone marrow, which provides stable, long-term hematopoiesis after transplantation. Rh-123 labels mitochondria with increasing intensity proportional to cellular activation, however the intensity of staining also correlates with the multidrug resistance (MDR) phenotype, as Rh-123 is a substrate for P-glycoprotein (P-gp). To address the mechanisms of long-term repopulating HSC discrimination by Rh-123, mouse bone marrow stem and progenitor cells were isolated based on surface antigen expression and subsequently separated into subsets using various fluorescent probes sensitive to mitochondrial characteristics and/or MDR function. We determined the cell cycle status of the separated populations and tested for HSC function using transplantation assays. Based on blocking studies using MDR modulators, we observed little efflux of Rh-123 from HSC obtained from young (3- to 4-week-old) mice, but significant efflux from HSC derived from older animals. A fluorescent MDR substrate (Bodipy-verapamil, BodVer) and Rh-123 both segregated quiescent cells into a dim-staining population, however Rh-123-based separations resulted in better enrichment of HSC function. Similar experiments using two other fluorescent probes with specificity for either mitochondrial mass or membrane potential indicated that mitochondrial activation is more important than either mitochondrial mass or MDR function in defining HSC in young mice. This conclusion was supported by morphologic studies of cell subsets separated by Rh-123 staining.

Natural proteoglycan receptor analogs determine the dynamics of Opa adhesin-mediated gonococcal infection of Chang epithelial cells.

Many bacterial pathogens possess a complex machinery for the induction and/or secretion of factors that promote their uptake by mammalian cells. We searched for the molecular basis of the 60- to 90-min lag time in the interaction of Neisseria gonorrhoeae carrying the heparin-binding Opa adhesin with Chang epithelial cells. Infection assays in the presence of chloramphenicol demonstrated that the Opa-mediated gonococcal infection of Chang cells required bacterial protein synthesis when the microorganisms were derived from GC agar but not when grown in liquid media. Further analysis indicated that contact with agar ingredients rather than the growth state of the microorganisms determined the infection dynamics. DEAE chromatography of GC agar extracts and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses and testing of collected fractions in infection assays identified negatively charged high-molecular-weight polysaccharides in the agar as inhibitors of the cellular infection. Electron microscopy showed that agar-grown gonococci were surrounded by a coat of alcian blue-positive material, probably representing accreted polysaccharides. Similar antiphagocytic material was isolated from bovine serum, indicating that in biological fluids gonococci producing the heparin-binding Opa adhesin may become covered with externally derived polysaccharides as well. Binding assays with gonococci and epithelial proteoglycan receptors revealed that polysaccharides derived from agar or serum compete with the proteoglycans for binding of the heparin-binding Opa adhesin and thus act as receptor analogs. Growth of gonococci in a polysaccharide-free environment resulted in optimal proteoglycan receptor binding and rapid bacterial entry into Chang cells. The recognition that gonococci with certain phenotypes can recruit surface polysaccharides that determine in vitro infection dynamics adds a different dimension to the well-recognized biological significance of genetic variation for this pathogen.

Expression of Aleutian mink disease parvovirus capsid proteins in defined segments: localization of immunoreactive sites and neutralizing epitopes to specific regions.

The capsid proteins of the ADV-G isolate of Aleutian mink disease parvovirus (ADV) were expressed in 10 nonoverlapping segments as fusions with maltose-binding protein in pMAL-C2 (pVP1, pVP2a through pVP2i). The constructs were designed to capture the VP1 unique sequence and the portions analogous to the four variable surface loops of canine parvovirus (CPV) in individual fragments (pVP2b, pVP2d, pVP2e, and pVP2g, respectively). The panel of fusion proteins was immunoblotted with sera from mink infected with ADV. Seropositive mink infected with either ADV-TR, ADV-Utah, or ADV-Pullman reacted preferentially against certain segments, regardless of mink genotype or virus inoculum. The most consistently immunoreactive regions were pVP2g, pVP2e, and pVP2f, the segments that encompassed the analogs of CPV surface loops 3 and 4. The VP1 unique region was also consistently immunoreactive. These findings indicated that infected mink recognize linear epitopes that localized to certain regions of the capsid protein sequence. The segment containing the hypervariable region (pVP2d), corresponding to CPV loop 2, was also expressed from ADV-Utah. An anti-ADV-G monoclonal antibody and a rabbit anti-ADV-G capsid antibody reacted exclusively with the ADV-G pVP2d segment but not with the corresponding segment from ADV-Utah. Mink infected with ADV-TR or ADV-Utah also preferentially reacted with the pVP2d sequence characteristic of that virus. These results suggested that the loop 2 region may contain a type-specific linear epitope and that the epitope may also be specifically recognized by infected mink. Heterologous antisera were prepared against the VP1 unique region and the four segments capturing the variable surface loops of CPV. The antisera against the proteins containing loop 3 or loop 4, as well as the anticapsid antibody, neutralized ADV-G infectivity in vitro and bound to capsids in immune electron microscopy. These results suggested that regions of the ADV capsid proteins corresponding to surface loops 3 and 4 of CPV contain linear epitopes that are located on the external surface of the ADV capsid. Furthermore, these linear epitopes contain neutralizing determinants. Computer comparisons with the CPV crystal structure suggest that these sequences may be adjacent to the threefold axis of symmetry of the viral particle.