Aleutian mink disease parvovirus infection of mink macrophages and human macrophage cell line U937: demonstration of antibody-dependent enhancement of infection.

Aleutian mink disease parvovirus (ADV) infects macrophages in adult mink. The virulent ADV-Utah I strain, but not the cell culture-adapted ADV-G strain, infects mink peritoneal macrophage cultures and the human macrophage cell line U937 in vitro. However, preincubation of ADV-G with ADV-infected mink serum enhanced its infectivity for U937 cells. the enhancing activity was present in the protein A-binding immunoglobulin G fraction in the serum, but F(ab')2 fragments failed to enhance the infection. On the other hand, the same sera inhibited ADV-G infection of Crandell feline kidney (CRFK) cells. Although U937 cells were not fully permissive for antibody-enhanced ADV-G infection, ADV mRNA expression, genome amplification, and protein expression were identical to those found previously for ADV-Utah I infection of U937 cells. Preincubation of ADV-Utah I with soluble protein A partly inhibited the infection of U937 cells but did not affect infection of CRFK cells. In mink peritoneal macrophages, preincubation with the infected mink serum did not make ADV-G infectious. However, the infectivity for mink macrophages of antibody-free ADV-Utah I prepared from the lungs of infected newborn mink kits was enhanced by ADV-infected mink serum. Moreover, protein A partly blocked ADV-Utah I infection of mink macrophage cultures. These results suggested that ADV-Utah I enters mink macrophages and U937 cells via an Fc receptor-mediated mechanism. This mechanism, antibody-dependent enhancement, may also contribute to ADV infection in vivo. Furthermore, since ADV infection in mink is characterized by overproduction of anti-ADV immunoglobulins, antibody-dependent enhancement may play a critical role in the establishment of persistent infection with ADV in vivo.     

Studies on the sequential development of acute interstitial pneumonia caused by Aleutian disease virus in mink kits.

We studied different parameters during the development of acute interstitial pneumonia in mink kits caused by neonatal infection with Aleutian disease virus (ADV). When histological lesions, presence of intranuclear inclusion bodies, and intranuclearly localized ADV antigen were correlated with levels of single-stranded virion and duplex replicative forms of ADV DNA in the different tissues, it was concluded that the lung, probably alveolar type II cells, is the major primary target for viral replication and cytopathology. The presence of the duplex dimeric replicative-form DNA, a strong marker of parvovirus replication, was also observed in low amount in the mesenteric lymph node, suggesting replication of ADV in this organ, although no viral cytopathology could be demonstrated. Moreover, a few intranuclear inclusion bodies were demonstrated in kidney and liver from affected kits, but intranuclearly localized ADV antigen could not be demonstrated in liver sections, and neither could duplex dimer replicative-form DNA, suggesting that these organs are nevertheless not a major site of ADV replication. When the data were compared with results previously reported for ADV-infected adult mink and ADV-infected permissive cell cultures, the data suggested that the pattern of ADV replication in alveolar type II cells is similar to that seen in infected cell cultures but that the replication in the other kit organs resembles the restricted pattern seen in adult mink.     

Analysis of Aleutian disease virus infection in vitro and in vivo: demonstration of Aleutian disease virus DNA in tissues of infected mink.

Aleutian disease virus (ADV) infection was analyzed in vivo and in vitro to compare virus replication in cell culture and in mink. Initial experiments compared cultures of Crandell feline kidney (CRFK) cells infected with the avirulent ADV-G strain or the highly virulent Utah I ADV. The number of ADV-infected cells was estimated by calculating the percentage of cells displaying ADV antigen by immunofluorescence (IFA), and several parameters of infection were determined. Infected cells contained large quantities of viral DNA (more than 10(5) genomes per infected cell) as estimated by dot-blot DNA-DNA hybridization, and much of the viral DNA, when analyzed by Southern blot hybridization, was found to be of a 4.8-kilobase-pair duplex monomeric replicative form (DM DNA). Furthermore, the cultures contained 7 to 67 fluorescence-forming units (FFU) per infected cell, and the ADV genome per FFU ratio ranged between 2 X 10(3) and 164 X 10(3). Finally, the pattern of viral antigen detected by IFA was characteristically nuclear, although cytoplasmic fluorescence was often found in the same cells. Because no difference was noted between the two virus strains when cultures containing similar numbers of infected cells were compared, it seemed that both viruses behaved similarly in infected cell culture. These data were used as a basis for the analysis of infection of mink by virulent Utah I ADV. Ten days after infection, the highest levels of viral DNA were detected in spleen (373 genomes per cell), mesenteric lymph node (MLN; 750 genomes per cell), and liver (373 genomes per cell). In marked contrast to infected CRFK cells, the predominant species of ADV DNA in all tissues was single-stranded virion DNA; however, 4.8-kilobase-pair DM DNA was found in MLN and spleen. This observation suggested that MLN and spleen were sites of virus replication, but that the DNA found in liver reflected sequestration of virus produced elsewhere. A final set of experiments examined MLN taken from nine mink 10 days after Utah I ADV infection. All of the nodes contained ADV DNA (46 to 750 genomes per cell), and although single-stranded virion DNA was always the most abundant species, DM DNA was observed. All of the lymph nodes contained virus infectious for CRFK cells, but when the genome per FFU ratio was calculated, virus from the lymph nodes required almost 1,000 times more genomes to produce an FFU than did virus prepared from infected cell cultures.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cytokine profiles in adult mink infected with Aleutian mink disease parvovirus

The aim of this study was to examine the levels of gamma interferon (IFN-gamma)-, interleukin 4 (IL-4)-, and IL-8-producing cells in peripheral blood mononuclear cells from mink infected with the Aleutian mink disease parvovirus (ADV). As expected, ADV-infected mink developed high plasma gamma globulin values (hypergammaglobulinemia) and enhanced quantities of CD8-positive (CD8(+)) cells in the blood during the infection. We quantified the percentages of IFN-gamma- and IL-4-positive lymphocytes and IL-8-positive monocytes up to week 38 after virus challenge. The results clearly indicated marked increases in the percentages of IFN-gamma- and IL-4-producing lymphocytes during ADV infection. The total number of IL-8-producing monocytes in the blood of ADV-infected mink stayed fairly constant during the infection. In order to characterize the phenotype of the cytokine-producing cells, we performed double-labeling fluorescence-activated cell sorter (FACS) experiments with CD8 surface labeling in one channel and cytokine intracellular staining in the other. We found that most IFN-gamma and IL-4 in ADV-infected mink was produced by CD8(+) cells, while in the uninfected mink, these cytokines were primarily produced by a cell type that was not CD8 (possibly CD4-positive cells). We also observed that IL-8 was almost exclusively produced by monocytes. All of the above findings led us to conclude that both Th1- and Th2-driven immune functions are found in mink plasmacytosis.     

The relationship between capsid protein (VP2) sequence and pathogenicity of Aleutian mink disease parvovirus (ADV): a possible role for raccoons in the transmission of ADV infections.

Aleutian mink disease parvovirus (ADV) DNA was identified by PCR in samples from mink and raccoons on commercial ranches during an outbreak of Aleutian disease (AD). Comparison of DNA sequences of the hypervariable portion of VP2, the major capsid protein of ADV, indicated that both mink and raccoons were infected by a new isolate of ADV, designated ADV-TR. Because the capsid proteins of other parvoviruses play a prominent role in the determination of viral pathogenicity and host range, we decided to examine the relationship between the capsid protein sequences and pathogenicity of ADV. Comparison of the ADV-TR hypervariable region sequence with sequences of other isolates of ADV revealed that ADV-TR was 94 to 100% related to the nonpathogenic type 1 ADV-G at both the DNA and amino acid levels but less than 90% related to other pathogenic ADVs like the type 2 ADV-Utah, the type 3 ADV-ZK8, or ADV-Pullman. This finding indicated that a virus with a type 1 hypervariable region could be pathogenic. To perform a more comprehensive analysis, the complete VP2 sequence of ADV-TR was obtained and compared with that of the 647-amino-acid VP2 of ADV-G and the corresponding VP2 sequences of the pathogenic ADV-Utah, ADV-Pullman, and ADV-ZK8. Although the hypervariable region amino acid sequence of ADV-TR was identical to that of ADV-G, there were 12 amino acid differences between ADV-G and ADV-TR. Each of these differences was at a position where other pathogenic isolates also differed from ADV-G. Thus, although ADV-TR had the hypervariable sequence of the nonpathogenic type 1 ADV-G, the remainder of the VP2 sequence resembled sequences of other pathogenic ADVs. Under experimental conditions, ADV-TR and ADV-Utah were highly pathogenic and induced typical AD in trios of both Aleutian and non-Aleutian mink, whereas ADV-Pullman was pathogenic only for Aleutian mink and ADV-G was noninfectious. Trios of raccoons experimentally inoculated with ADV-TR and ADV-Utah all became infected with ADV, but only a single ADV-Pullman-inoculated raccoon showed evidence of infection. Furthermore, none of the ADV isolates induced pathological findings of AD in raccoons. Finally, when a preparation of ADV-TR prepared from infected raccoon lymph nodes was inoculated into mink and raccoons, typical AD was induced in Aleutian and non-Aleutian mink, but raccoons failed to show serological or pathological evidence of infection. These results indicated that raccoons can become infected with ADV and may have a role in the transmission of virus to mink but that raccoon-to-raccoon transmission of ADV is unlikely.     

In situ molecular hybridization for detection of Aleutian mink disease parvovirus DNA by using strand-specific probes: identification of target cells for viral replication in cell cultures and in mink kits with virus-induced interstitial pneumonia.

Strand-specific hybridization probes were utilized in in situ molecular hybridization specifically to localize replicative form DNA of Aleutian mink disease parvovirus (ADV). Throughout in vitro infection, duplex replicative form DNA of ADV was located in the cell nuclei. Single-stranded virion DNA and capsid proteins were present in the nuclei early in infection, but were later translocated to the cytoplasm. In neonatal mink, ADV causes acute interstitial pneumonia, and replicative forms of viral DNA were found predominantly in alveolar type II cells of the lung. Viral DNA was also found in other organs, but strand-specific probes made it possible to show that most of this DNA represented virus sequestration. In addition, glomerular immune complexes containing intact virions were detected, suggesting that ADV virions may have a role in the genesis of ADV-induced glomerulonephritis.     

Analysis of experimental mink enteritis virus infection in mink: in situ hybridization, serology, and histopathology.

Strand-specific hybridization probes were used in in situ hybridization studies to localize cells containing mink enteritis virus (MEV) virion DNA or MEV replicative-form DNA and mRNA. Following the experimental MEV infection of 3-month-old unvaccinated mink, a significant increase in serum antibodies to MEV was detected at postinfection day (PID) 6, 2 days after the onset of fecal shedding of virus. Prior to the appearance of virus in feces, viral DNA could be detected in the mesenteric lymph node and intestine. The largest percentage of cells positive for virion DNA was 10% and was detected in the intestine on PID 6. However, replication of the virus apparently peaked at PID 4. The number of MEV replicative-form DNA and mRNA molecules was found to be approximately 250,000 copies per infected lymph node cell or crypt epithelial cell. The localization, levels, and time course of viral replication have important implications for the pathogenesis of MEV-induced disease. The data presented on MEV are correlated with earlier results on the other mink parvovirus, Aleutian mink disease parvovirus, and a possible explanation for the remarkable differences in pathogenesis of disease caused by these two parvoviruses is discussed.     

Monoclonal antibodies against Aleutian disease virus distinguish virus strains and differentiate sites of virus replication from sites of viral antigen sequestration.

Monoclonal antibodies (mAbs) were used to study antigenic differences among strains of Aleutian disease virus (ADV) and to characterize viral proteins in vitro and in vivo. A number of ADV field strains could be discriminated, and highly virulent Utah I ADV was clearly delineated from the tissue culture-adapted avirulent ADV-G strain. This specificity could be demonstrated by indirect immunofluorescence against infected cultures of Crandell feline kidney cells or against tissues of Utah I ADV-infected mink. Viral antigens were demonstrated in both the nuclei and the cytoplasm of infected tissue culture cells. However, in mink mesenteric lymph node, spleen, and liver, viral antigen was observed only in the cytoplasm. Absence of nuclear fluorescence suggested that the detected antigen represented phagocytized viral antigens rather than replicating virus. This conclusion was supported by the finding that mAbs reactive only against low-molecular-weight polypeptides derived from intact viral proteins gave the same pattern of in vivo fluorescence as mAbs with broad reactivity for large or small (or both) viral polypeptides. The distribution of infected cells was the same as that described for macrophages in these tissues and suggested that cells of the reticuloendothelial system had sequestered viral antigens.     

Investigation of the pathogenesis of transplacental transmission of Aleutian mink disease parvovirus in experimentally infected mink.

The transplacental transmission of Aleutian mink disease parvovirus (ADV) was studied in experimental infection of 1-year-old female non-Aleutian mink. The ADV-seronegative female mink were inoculated with ADV prior to mating or after the expected implantation of the embryos during pregnancy. A group of uninfected females served as a control group. Animals from each group were killed prior to or shortly after parturition. The in situ hybridization technique with radiolabeled strand-specific RNA probes was used to determine target cells of virus infection and virus replication. In both infected groups, ADV crossed the endotheliochorial placental barrier, although animals infected before mating already had high antibody titers against ADV at the time of implantation. The percentage of dead and resorbed fetuses was much higher in dams infected before mating. In the placentae of these mink, virus DNA and viral mRNA were detected in cells in the mesenchymal stroma of the placental labyrinth and hematoma but only occasionally in the cytotrophoblast of the placental hematoma. Placentae of animals infected during pregnancy showed in addition very high levels of virus and also viral replication in a large number of cytotrophoblast cells in the placental hematoma, which exhibited distinct inclusion bodies. In both groups, neither virus nor virus replication could be detected in maternal endothelial cells or fetal syncytiotrophoblast of the placental labyrinth. Fetuses were positive for virus and viral replication at high levels in a wide range of tissues. Possible routes of transplacental transmission of ADV and the role of trophoblast cells as targets for viral replication are discussed.     

Identification of a cell surface protein from Crandell feline kidney cells that specifically binds Aleutian mink disease parvovirus

Aleutian mink disease parvovirus (ADV) is the etiological agent of Aleutian disease of mink. The acute disease caused by ADV consists of permissive infection of alveolar type II cells that results in interstitial pneumonitis. The permissive infection is experimentally modeled in vitro by infecting Crandell feline kidney (CrFK) cells with a tissue culture-adapted isolate of ADV, ADV-G. ADV-G VP2 empty virions expressed in a recombinant baculovirus system were analyzed for the ability to bind to the surface of CrFK cells. Radiolabeled VP2 virions bound CrFK cells specifically, while they did not bind either Mus dunni or Spodoptera frugiperda cells, cells which are resistant to ADV infection. The binding to CrFK cells was competitively inhibited by VP2 virions but not by virions of cowpea chlorotic mottle virus (CCMV), another unenveloped virus similar in size to ADV. Furthermore, preincubation of CrFK cells with the VP2 virions blocked infection by ADV-G. The VP2 virions were used in a virus overlay protein binding assay to identify a single protein of approximately 67 kDa, named ABP (for ADV binding protein), that demonstrates specific binding of VP2 virions. Exogenously added VP2 virions were able to competitively inhibit the binding of labeled VP2 virions to ABP, while CCMV virions had no effect. Polyclonal antibodies raised against ABP reacted with ABP on the outer surface of CrFK cells and blocked infection of CrFK cells by ADV-G. In addition, VP2 virion attachment to CrFK cells was blocked when the VP2 virions were preincubated with partially purified ABP. Taken together, these results indicate that ABP is a cellular receptor for ADV.     

Proteins of viral and cellular origin bind to the Aleutian disease virus (ADV) DNA 3'-terminal hairpin: presentation of a scheme for encapsidation of ADV DNA.

We have observed the binding of viral and cellular proteins to the Aleutian disease virus (ADV) 3' terminus of replicative-form DNA. Gel retardation assays showed specific band shifts produced by whole-cell extracts from either ADV-infected or uninfected cells, as well as band reduction produced by ADV capsids. In all cases, binding was confined to the turnaround, T-shaped terminal form; no binding to the extended conformation of replicative-form DNA was detected. This indicates the importance of the T-shaped secondary structure in protein recognition. We have previously reported the binding of a 3'-terminal ADV DNA restriction fragment to the ADV capsid protein VP1 (K. Willwand and O.-R. Kaaden, Virology 166:52-57, 1988). Here we show that the region between nucleotides 14 and 102 on the ADV genome is required for binding. It is suggested that the VP1-DNA interaction mediates the binding of ADV DNA to empty viral capsids and that this is followed by displacement synthesis and packaging of ADV progeny DNA. A scheme for the possible mechanism of this process is presented.     

Characterization of chimeric full-length molecular clones of Aleutian mink disease parvovirus (ADV): identification of a determinant governing replication of ADV in cell culture.

The ADV-G strain of Aleutian mink disease parvovirus (ADV) is nonpathogenic for mink but replicates permissively in cell culture, whereas the ADV-Utah 1 strain is highly pathogenic for mink but replicates poorly in cell culture. In order to relate these phenotypic differences to primary genomic features, we constructed a series of chimeric plasmids between a full-length replication-competent molecular clone of ADV-G and subgenomic clones of ADV-Utah 1 representing map units (MU) 15 to 88. After transfection of the plasmids into cell culture and serial passage of cell lysates, we determined that substitution of several segments of the ADV-Utah 1 genome (MU 15 to 54 and 65 to 73) within an infectious ADV-G plasmid did not impair the ability of these constructs to yield infectious virus in vitro. Like ADV-G, the viruses derived from these replication-competent clones caused neither detectable viremia 10 days after inoculation nor any evidence of Aleutian disease in adult mink. On the other hand, other chimeric plasmids were incapable of yielding infectious virus and were therefore replication defective in vitro. The MU 54 to 65 EcoRI-EcoRV fragment of ADV-Utah 1 was the minimal segment capable of rendering ADV-G replication defective. Substitution of the ADV-G EcoRI-EcoRV fragment into a replication-defective clone restored replication competence, indicating that this 0.53-kb portion of the genome, wholly located within shared coding sequences for the capsid proteins VP1 and VP2, contained a determinant that governs replication in cell culture. When cultures of cells were studied 5 days after transfection with replication-defective clones, rescue of dimeric replicative form DNA and single-stranded progeny DNA could not be demonstrated. This defect could not be complemented by cotransfection with a replication-competent construction.     

Replication of Aleutian mink disease parvovirus in vivo is influenced by residues in the VP2 protein.

Aleutian mink disease parvovirus (ADV) is the etiological agent of Aleutian disease of mink. Several ADV isolates have been identified which vary in the severity of the disease they elicit. The isolate ADV-Utah replicates to high levels in mink, causing severe Aleutian disease that results in death within 6 to 8 weeks, but does not replicate in Crandell feline kidney (CrFK) cells. In contrast, ADV-G replicates in CrFK cells but does not replicate in mink. The ability of the virus to replicate in vivo is determined by virally encoded determinants contained within a defined region of the VP2 gene (M. E. Bloom, J. M. Fox, B. D. Berry, K. L. Oie, and J. B. Wolfinbarger. Virology 251:288-296, 1998). Within this region, ADV-G and ADV-Utah differ at only five amino acid residues. To determine which of these five amino acid residues comprise the in vivo replication determinant, site-directed mutagenesis was performed to individually convert the amino acid residues of ADV-G to those of ADV-Utah. A virus in which the ADV-G VP2 residue at 534, histidine (H), was converted to an aspartic acid (D) of ADV-Utah replicated in CrFK cells as efficiently as ADV-G. H534D also replicated in mink, causing transient viremia at 30 days postinfection and a strong antibody response. Animals infected with this virus developed diffuse hepatocellular microvesicular steatosis, an abnormal accumulation of intracellular fat, but did not develop classical Aleutian disease. Thus, the substitution of an aspartic acid at residue 534 for a histidine allowed replication of ADV-G in mink, but the ability to replicate was not sufficient to cause classical Aleutian disease.     

Restriction analysis of the DNA of aleutian mink disease virus strain P-1

The II-1 strain of the Aleutian disease virus (ADV-II-1) was isolated from experimentally infected mink organs. The viral particles were isolated having 23 to 24 nm in diameter with the buoyant density of the virions in CsCl gradient being 1.41 g.ml-1. The single stranded ADV DNA extracted from the purified virus particles had the molecular mass about 1.4 . 10(6) (4800 bases). The double-stranded replicative form of ADV DNA has been synthesized in vitro with the use of a large "Klenow" fragment of DNA-polymerase I. A restriction endonuclease map of ADV-II-1 DNA has been constructed with the use of in vitro synthesized double-stranded DNA.     

Caspase cleavage of the nonstructural protein NS1 mediates replication of Aleutian mink disease parvovirus

Virus-induced apoptosis of infected cells can limit both the time and the cellular machinery available for virus replication. Hence, many viruses have evolved strategies to specifically inhibit apoptosis. However, Aleutian mink disease parvovirus (ADV) is the first example of a DNA virus that not only induces apoptosis but also utilizes caspase activity to facilitate virus replication. To determine the function of caspase activity during ADV replication, virus-infected cell lysates or purified ADV proteins were incubated with various purified caspases. Caspases cleaved the major nonstructural protein of ADV (NS1) at two caspase recognition sequences, whereas ADV structural proteins could not be cleaved. Importantly, the NS1 products could be identified in ADV-infected cells but were not present in infected cells pretreated with caspase inhibitors. By mutating putative caspase cleavage sites (D to E), we mapped the two cleavage sites to amino acid residues NS1:227 (INTD downward arrow S) and NS1:285 (DQTD downward arrow S). Replication of ADV containing either of these mutations was reduced 10(3)- to 10(4)-fold compared to that of wild-type virus, and a construct containing both mutations was replication defective. Immunofluorescent studies revealed that cleavage was required for nuclear localization of NS1. The requirement for caspase activity during permissive replication suggests that limitation of caspase activation and apoptosis in vivo may be a novel approach to restricting virus replication.     

Molecular comparisons of in vivo- and in vitro-derived strains of Aleutian disease of mink parvovirus.

DNA from one cell culture-adapted and two pathogenic strains of Aleutian disease of mink parvovirus (ADV) was molecularly cloned into the vectors pUC18 and pUC19. The DNA from the two pathogenic strains (ADV-Utah I and ADV-Pullman) was obtained from virus purified directly from the organs of infected mink, whereas the DNA from the nonpathogenic ADV-G was derived from cell culture material. The cloned segment from all three viruses represented a 3.55-kilobase-pair BamHI (15 map units) to HindIII (88 map units) fragment. Detailed physical mapping studies indicated that all three viruses shared 29 of 46 restriction endonuclease recognition sites but that 6 sites unique to the pathogenic strains and 5 sites unique to ADV-G were clustered in the portion of the genome expected to code for structural proteins. Clones from all three viruses directed the synthesis of two ADV-specific polypeptides with molecular weights of approximately 57 and 34 kilodaltons. Both species reacted with sera from infected mink as well as with a monoclonal antibody specific for ADV structural proteins. Because production of these ADV antigens was detected in both pUC18 and pUC19 and was not influenced by isopropyl-beta-D-thiogalactopyranoside (IPTG) induction, their expression was not regulated by the lac promoter of the pUC vector, but presumably by promoterlike sequences found within the ADV DNA. The proteins specified by the clones of ADV-G were 2 to 3 kilodaltons smaller than those of the two pathogenic strains, although the DNA segments were identical in size. This difference in protein molecular weights may correlate with pathogenicity, because capsid proteins of pathogenic and nonpathogenic strains of ADV exhibit a similar difference.     

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.