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

Abstract

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 replicationcompetent 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.

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

“…The following preparations of ADV were used in this study. (i) The nonpathogenic type 1 ADV-G strain was derived from a molecularly cloned stock of virus (XXI-Q-3-15) (19). (ii) The type 2 ADV-Utah 1 strain, prepared from acutely infected adult mink, was derived from the same preparation used in previous studies at Rocky Mountain Laboratories (6,7,23,31,42); for convenience, it is referred to herein simply as ADV-Utah.…”

“…Plasmids pXXI-Q-3-15 (containing the entire ADV-G sequence) (16,19) and pXX-B-1 (a full-length plasmid substituting the ADV-Utah sequence for the segment from map units 15 to 88) (19) were used to provide templates of known sequence for PCR. Additional molecular clones of ADV-Utah, designated ADV-Utah kit, were derived from duplex replicative-form DNA (21) from the lungs of ADV-Utah-infected mink kits (6,16,19,21,27). The sequences for ADV-Utah and the Danish type 3 virus isolated from ADV-K (designated ADV-ZK8) are published (16,18,19,37).…”

“…ADV-Pullman, on the other hand, induces severe disease in Aleutian mink, but its pathogenicity is greatly reduced in non-Aleutian animals (23,42,43,64). Furthermore, the cell culture-adapted ADV-G strain has a severely reduced capacity for in vivo replication and is nonpathogenic, although it replicates permissively in cell culture (19,24). These findings point out that for ADV infections, both the viral strain and the genotype of the host are important in determining the disease picture.…”

“…Molecular virology studies have begun to elucidate genomic features that may influence pathogenicity and host range for isolates of ADV (16,18,19). The complete sequence and transcription map of the 4,748-nucleotide (nt) nonpathogenic ADV-G isolate of ADV are known (4,16,18).…”

“…600-bp EcoRV-BstEII fragment within the capsid protein gene (map units 64 to 65) (20,37,38). Definition of the hypervariable region has led to a preliminary typing scheme based on this sequence (18,19,38). For example, the nonpathogenic cell culture-adapted strain, ADV-G, is defined as type 1, whereas the highly pathogenic ADV-Utah strain is designated type 2 (38), and the various sequence types appear to be quite stable (37).…”