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)     

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.     

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.     

Grouping of adenoviruses and identification of restriction endonuclease cleavage patterns of adenovirus DNAs using infected cell DNA: simple and practical methods

Simple and practical methods for grouping of adenoviruses and for identification of restriction endonuclease cleavage patterns of viral DNA were established by using infected cell DNA. DNA homology groupings of adenoviruses could be examined by spot hybridization, and restriction endonuclease cleavage patterns of viral DNAs could be obtained by Southern blot hybridization, by using infected cell DNA. The method was very sensitive and allowed the identification of the cleavage pattern of viral DNA of the inoculum by means of cell DNA extracted from infected cells with undetectable cytopathic effect (CPE). In ethidium bromide-stained gels without Southern blot hybridization, the restriction endonuclease cleavage pattern of viral DNA could be detected precisely in spite of background staining due to cellular DNA. The preparation of infected cell DNA used in these procedures was technically much easier than that of viral DNA. These methods require only a small number of infected cells and allow many isolates to be investigated with ease.     

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.     

Molecular cloning of the Aleutian disease virus genome: expression of Aleutian disease virus antigens by a recombinant plasmid

Three nonoverlapping segments representing approximately 80% of the 4.8-kilobase pair Aleutian disease virus (ADV-G) duplex genome were molecularly cloned into either bacteriophage M13mp9 (M13bm2 = 0.07 to 0.15 map unit; M13bm1 = 0.15 to 0.54 map unit) or plasmid pUC8 (pBM1 = 0.54 to 0.88 map units). In addition the 0.54- to 0.88-map unit segment of a Danish isolate of ADV (DK ADV) was also cloned into pUC8 (pBM2). The recombinant plasmids pBM1 and pBM2 induced expression of several polypeptides in Escherichia coli JM103 that were specifically recognized by sera from mink infected with ADV. The same three proteins with approximate molecular weights of 55,000, 34,000, and 27,000 were detected both by immune blotting and by immunoprecipitation of [35S]methionine-labeled JM103 (pBM1). None of these proteins were recognized in JM103 or JM103 (pUC8), nor were they detected by sera from normal mink. Purified pBM1 and pBM2 DNA appeared identical in size by gel analysis and contour length measurement, and electron microscopic heteroduplex mapping revealed no visible areas of heterology. However, restriction endonuclease mapping showed that pBM2 was different from pBM1, indicating that this segment of the ADV genome was similar but not identical for two strains of ADV (ADV-G and DK ADV). Furthermore, when cloned DNA from ADV-G was labeled with [32P]dCTP by nick translation, DNA relatedness to several field strains of ADV (Utah I, Pullman, and DK), but not to mink enteritis virus or cellular DNA, was shown by Southern blot hybridization.     

Role of alveolar type II cells and of surfactant-associated protein C mRNA levels in the pathogenesis of respiratory distress in mink kits infected with Aleutian mink disease parvovirus.

Neonatal mink kits infected with Aleutian mink disease parvovirus (ADV) develop an acute interstitial pneumonia with clinical symptoms and pathological lesions that resemble those seen in preterm human infants with respiratory distress syndrome and in human adults with adult respiratory distress syndrome. We have previously suggested that ADV replicates in the alveolar type II epithelial cells of the lung. By using double in situ hybridization, with the simultaneous use of a probe to detect ADV replication and a probe to demonstrate alveolar type II cells, we now confirm this hypothesis. Furthermore, Northern (RNA) blot hybridization showed that the infection caused a significant decrease of surfactant-associated protein C mRNA produced by the alveolar type II cells. We therefore suggest that the severe clinical symptoms and pathological changes characterized by hyaline membrane formation observed in ADV-infected mink kits are caused by a dysfunction of alveolar surfactant similar to that observed in respiratory distress syndrome in preterm infants. However, in the infected mink kits the dysfunction is due to the replication of ADV in the lungs, whereas the dysfunction of surfactant in preterm infants is due to lung immaturity.     

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.     

Construction of an infectious molecular clone of the autonomous parvovirus minute virus of mice.

The linear single-stranded DNA genome of minute virus of mice, an autonomous parvovirus, was cloned in duplex form into the bacterial plasmid pBR322. The recombinant clones of minute virus of mice were infectious when transfected into monolayers of human 324K cells and produced virus plaques with an efficiency of about 6% that obtained with duplex replicative-form DNA purified from cells infected with minute virus of mice. Southern blot analysis of transfected cells indicated that the cloned minute virus of mice genome requires both termini to be intact for excision and replication as a linear duplex molecule.     

In vitro synthesis of double-stranded DNA from the Kilham rat virus single-stranded DNA genome.

Double-stranded, full-length linear DNA was synthesized in vitro by using single-stranded linear DNA as a self-priming template from the parvovirus Kilham rat virus and Escherichia coli DNA polymerase "large fragment" as the polymerizing enzyme. To ascertain the order of the synthesis of the cleavage fragments and to assess the accuracy of the in vitro synthesis, restriction endonuclease cleavage sites with known recognition sequences were mapped on the DNA. Comparing the cleavage pattern of the synthesized DNA with that of double-stranded viral DNA isolated from infected cells confirms that the in vitro synthesis produces a faithful copy of the viral single-stranded genome. Electron micrographs of the in vitro product reveal it to be a double-stranded linear molecule.     

Characterization of the protein and nucleic acid of Aleutian disease virus.

Aleutian disease virus (ADV) was extracted and purified from infected mink. Nucleic acid extracted from the virus was examined in an electron microscope. Three different sizes of molecule, with approximate lengths of 1.2, 0.55, and 0.25 micron, were observed. The ratios of the large molecules to the small molecules were similar in all the particles prepared under different conditions. Equilibrium CsCl density gradient centrifugation showed that ADV nucleic acid had a buoyant density of 1.733 g/cm3. In Cs2SO4, ADV had a lower buoyant density than that of double-stranded RNA. These properties and its sensitivity to DNase suggested that ADV contains DNA. Thermal denaturation curves revealed that the DNA of ADV had a single-stranded configuration. Polypeptide analysis of ADV by polyacrylamide gel electrophoresis revealed the presence of four polypepties, with molecular weights of 30,000, 27,000, 20,500, and 14,000. These polypeptides were present in a ratio of 10:3:10:1, respectively. The data suggested that ADV is closely related to the members of the parvovirus groups.     

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.     

Construction and characterization of new coliphage M13 cloning vectors

New single-stranded DNA cloning vectors have been constructed by the insertion of additional DNA fragments into a HaeII restriction site in the bacteriophage M13 duplex replicative form (RF). These inserts into the M13 genome bring a single restriction sites useful for cloning, including PstI, XorII, EcoRI, SstI, XhoI, KpnI, and PvuII. Drug-resistance genes cloned into M13 include the beta-lactamase (bla) gene and the chloramphenicol acetyl transferase (cat) gene. These vectors provide a convenient means of easily obtaining the separated strands of a cloned duplex DNA fragment by cloning the fragment in each of the two possible orientations. Standard cloning techniques commonly applied to double-stranded DNAs can be utilized to insert foreign DNAs into the duplex RF DNAs of these vectors. Cells transformed by chimeric DNAs extrude filamentous phage particles carrying a circular single-stranded copy of the chimeric viral strand. Because M13-infected cells continue to grow and divide, cells can be transformed to yield either plaques or drug-resistant colonies. Specific inserts are readily detected by plaque hybridization techniques using an appropriate probe. Chimeric viral single strands from virus particles in the supernatant of small volumes of infected cultures can be rapidly and sensitively analyzed by agarose gel electrophoresis to determine the size of an insert.     

Construction of replication-competent Herpesvirus saimiri deletion mutants

DNA fragments derived from the left end of Herpesvirus saimiri 11 L-DNA were cloned in Escherichia coli by using vector pBR322. Deletions were introduced within a cloned 7.4-kilobase-pair sequence by using restriction endonucleases that cut once or twice within this sequence. Permissive owl monkey kidney-cultured cells were transfected with parental strain 11 viral DNA plus cloned DNA with specific sequences deleted. By screening the progeny of these transfections with a limiting-dilution spot hybridization assay, we isolated recombinant viruses containing deletions in this region. A contiguous 4.5-kilobase-pair sequence representing 4.1% of the coding capacity of the virus was found to be unnecessary for virus replication in cultured cells. These deletion mutants will allow us to test whether sequences in this region are required for the lymphoma-inducing capacity of H. saimiri. These same procedures should also allow us to introduce foreign DNA sequences into this region for studying their expression.     

Aleutian mink disease parvovirus infection of mink peritoneal macrophages and human macrophage cell lines.

Aleutian mink disease parvovirus (ADV) mRNAs are found in macrophages in lymph nodes and peritoneal exudate cells from ADV-infected mink. Therefore, we developed an in vitro infection system for ADV by using primary cultures of mink macrophages or macrophage cell lines. In peritoneal macrophage cultures from adult mink, virulent ADV-Utah I strain showed nuclear expression of viral antigens with fluorescein isothiocyanate-labeled ADV-infected mink serum, but delineation of specific viral proteins could not be confirmed by immunoblot analysis. Amplification of ADV DNA and production of replicative-form DNA were observed in mink macrophages by Southern blot analysis; however, virus could not be serially propagated. The human macrophage cell line U937 exhibited clear nuclear expression of viral antigens after infection with ADV-Utah I but not with tissue culture-adapted ADV-G. In U937 cells, ADV-Utah I produced mRNA, replicative-form DNA, virion DNA, and structural and nonstructural proteins; however, virus could not be serially passaged nor could [3H]thymidine-labeled virions be observed by density gradient analysis. These findings indicated that ADV-Utah I infection in U937 cells was not fully permissive and that there is another restricted step between gene amplification and/or viral protein expression and production of infectious virions. Treatment with the macrophage activator phorbol 12-myristate 13-acetate after adsorption of virus reduced the frequency of ADV-positive U937 cells but clearly increased that of human macrophage line THP-1 cells. These results suggested that ADV replication may depend on conditions influenced by the differentiation state of macrophages. U937 cells may be useful as an in vitro model system for the analysis of the immune disorder caused by ADV infection of macrophages.     

Subcellular localization of Aleutian mink disease parvovirus proteins and DNA during permissive infection of Crandell feline kidney cells.

Confocal microscopy allowed us to localize viral nonstructural (NS) and capsid (VP) proteins and DNA simultaneously in cells permissively infected with Aleutian mink disease parvovirus (ADV). Early after infection, NS proteins colocalized with viral DNA to form intranuclear inclusions, whereas VP proteins formed hollow intranuclear shells around the inclusions. Later, nuclei had irregular outlines and were virtually free of ADV products. In these cells, inclusions of viral DNA with or without associated NS protein were embedded in cytoplasmic VP protein. These findings implied that ADV replication within an infected cell is regulated spatially as well as temporally.     

Classification of herpesvirus saimiri into three groups based on extreme variation in a DNA region required for oncogenicity.

The leftmost 7 kilobase pairs of unique sequence L-DNA of herpesvirus saimiri was found to be highly variable among different strains as determined by restriction endonuclease analysis and blot hybridization. This region in one group of viruses (group A) showed only very weak hybridization with the DNA of two other groups. Similarly, a fragment of group B hybridized to DNA of its own group much more strongly than to group A. No homology was detected within a 1.2-kilobase-pair region between strain 11 (group A virus) and strain SMHI (group B) even under reduced stringency, and the adjacent 5.5-kilobase-pair segment of the region showed only a very weak intergroup hybridization. DNA of a third group of viruses (non-A, non-B) did not hybridize significantly with cloned fragments representing the leftmost 7-kilobase-pair region of either group A or group B. Since sequences in the highly variable region are required for the oncogenicity of the virus, these results raise interesting questions regarding the origin and function of this region of the genome.