Two techniques performed on skin biopsy samples (ear notches), immunohistochemistry (IHC) and antigen-capture ELISA (AgELISA), were compared for detection of bovine viral diarrhea virus (BVDV) persistent infection (PI) in 559 Angus calves between the ages of 1 and 5 months. The calves also were tested for BVDV infection using virus isolation (VI) and reverse transcription (RT)-PCR on buffy coat samples and for antibodies to BVDV types la and 2 by serum neutralization (SN). Sixty-seven of 559 (12.0%) calves tested positive at initial screening by IHC, AgELISA, or VI, and all 67 were kept for a minimum of 3 months and retested monthly by IHC, AgELISA, VI, RT-PCR, and SN. Of the calves positive at initial screening, 59/67 (88.1%) were determined PI and 8/67 (11.9%) were determined acutely infected. Both IHC and AgELISA detected 100% of PI calves; however, IHC and AgELISA also detected 6 and 8 acutely infected calves, respectively, at initial screening. Furthermore, IHC and AgELISA continued to detect 3 and 4 acutely infected calves, respectively, 3 months after initial screening. Three acutely infected calves had IHC staining indistinguishable from PI calves at initial screening. Both IHC and AgELISA are accurate at detecting BVDV-infected calves, but veterinarians and producers should be advised that both tests detect some calves acutely infected with BVDV in addition to PI animals. Repeat testing using VI or RT-PCR on buffy coat samples should be performed at 30 days after initial screening to conclusively discriminate between acute and PI.

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The 293 cell line that was generated by transforming human embryonic kidney cells with human adenovirus type 5 (HAV5) early region 1 (E1) sequences is an excellent host for generating and growing HAV5 recombinants with E1 deleted, but it does not support the replication of bovine adenovirus type 3 (BAV3). Madin-Darby bovine kidney (MDBK), an established bovine cell line, is an excellent host for growing and plaquing BAV3. For the purpose of combining the unique characteristics of these two cell lines (293 and MDBK), we generated a number of bovine ؋ human hybrid (BHH) cell lines. Comparison of three BHH hybrid clones-BHH3, BHH8, and BHH2C-with 293-Puro (puromycin-resistant 293 cells) and MDBK-Neo (G418-resistant MDBK cells) cell lines for total cellular DNA content, species-specific surface markers, isoenzyme analysis, and karyotyping indicate that they are hybrid in nature. BHH clones constitutively expressed the E1 proteins (E1A, E1B-21kDa, and E1B-55kDa) of HAV5 and efficiently supported the replication of both wild-type and replication-incompetent bovine or human adenoviruses. Transient gene expression experiments with a plasmid encoding the bacterial ␤-galactosidase gene demonstrated that BHH cell hybrids seem to have better transfection efficiencies than either of the parental cell lines. These cell lines will be useful for isolating and growing replication-competent human or bovine adenovirus recombinants with E1 deleted and for the study of cellular or viral factors important for viral replication. The development of somatic cell hybrids appears to be a simple way of combining some of the desirable characteristics present separately in two parental cell lines.The most widespread use of somatic cell hybrids is the formation of hybridomas for monoclonal antibody production. They are generated by the fusion of antibody-producing lymphocytes from a mouse or rat immunized with an antigen of interest with a myeloma cell line (1, 29). Production of interand intraspecific cell hybrids by means of polyethylene glycol (PEG)-induced fusion for extending the life of mammalian cells has been demonstrated (42). Somatic cell hybrids have also been used as tools for studying the chromosomal assignment of genes (12,26,27), mapping normal as well as tumorrelated genes (47), and localizing the site-specific integration of adeno-associated proviral DNA into human chromosome 19 (31, 49). Hybridization between bovine ϫ rodent, mouse, or hamster were exploited for the mapping of bovine genes (51), and bovine ϫ mouse hybrid cells were used for studying the replication of mengo virus (7,30,55).Adenoviral early region 1 (E1) genes are the first set of the genes that are expressed and are necessary for virus replication (22, 50). Adenovirus recombinants with E1 deleted are routinely produced and grown in a permissive cell line that constitutively expresses E1 proteins (17). The 293 cell line constitutively expresses E1 proteins and was derived from human embryonic kidney cells after transformation with human adenovirus type 5 (HA...

Like human adenovirus type 5 (HAV5), bovine adenovirus type 3 (BAV3) early region 1 (E1) consists of E1A and E1B transcriptional units. In order to characterize BAV3 E1 proteins and to isolate a cell line of bovine origin that expresses BAV3 E1, polyclonal antibodies specific to E1A, E1B-157R, and E1B-420R were raised in rabbits. BAV3 E1A, E1B-157R, and E1B-420R were identified as 40, 17, and 47 kDa proteins, and had a half-life of 45-60 min, and 4-6 and 4-6 h, respectively. It appeared that E1A and E1B-157R were phosphorylated at the serine/threonine residues, whereas, E1B 420R was phosphorylated at both the serine/threonine and tyrosine residues. Three cell lines, MDBK-221 (Madin Darby bovine kidney (MDBK) transfected with BAV3 E1), FBK-34 (primary fetal bovine kidney (FBK) cells transfected BAV3 E1), and FBRT-HE1 (bovine fetal retinal (FBRT) cells transfected with HAV5 E1) were isolated and characterized for E1 expression. FBK-34 or FBRT-HE1 supported the replication of an E1A-deleted BAV3 (BAV3DeltaE1AE3) to approximately 1-2 x 10(8) PFU/ml, whereas, the virus titers in MDBK-221 were approximately 10(7) PFU/ml. These cell lines will be useful in generating and growing BAV3 E1-deleted recombinants, and also for studying E1 protein interactions with a number of cellular and/or viral proteins.

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