Comparison of electron microscopic techniques for enumeration of endogenous retrovirus in mouse and Chinese hamster cell lines used for production of biologics.

Murine myeloma and Chinese hamster ovary cells are used widely in the manufacture of recombinant proteins for biopharmaceuticals. However, rodent cell lines express endogenous retrovirus, which necessitates appropriate design of purification processes to remove virus in excess of the calculated maximum retroviral load. Currently, electron microscopy is the method of choice for determination of retroviral titre in bulk harvest. In this study we compared three electron microscopy techniques to determine retroviral titre in bulk harvest. These were direct negative stain, negative stain after sucrose-density purification and thin section electron microscopy of pelleted supernatant. The study demonstrated that the level of C-type retrovirus associated with cells was predictive of the viral load in cell culture supernatants. The most accurate method for quantifying viral load was direct counting, followed by thin section of pelleted supernatant and negative stain after sucrose concentration. The most practical method was thin section of resuspended pelleted supernatant, which gave improved detection limits.

Xer-mediated site-specific recombination at cer generates Holliday junctions in vivo.

Normal segregation of the Escherichia coli chromosome and stable inheritance of multicopy plasmids such as ColE1 requires the Xer site-specific recombination system. Two putative lambda integrase family recombinases, XerC and XerD, participate in the recombination reactions. We have constructed an E. coli strain in which the expression of xerC can be tightly regulated, thereby allowing the analysis of controlled recombination reactions in vivo. Xer-mediated recombination in this strain generates Holliday junction-containing DNA molecules in which a specific pair of strands has been exchanged in addition to complete recombinant products. This suggests that Xer site-specific recombination utilizes a strand exchange mechanism similar or identical to that of other members of the lambda integrase family of recombination systems. The controlled in vivo recombination reaction at cer requires recombinase and two accessory proteins, ArgR and PepA. Generation of Holliday junctions and recombinant products is equally efficient in RuvC- and RuvC+ cells, and in cells containing a multicopy RuvC+ plasmid. Controlled XerC expression is also used to analyse the efficiency of recombination between variant cer sites containing sequence alterations and heterologies within their central regions.

Production and characterization of a monoclonal antibody recognizing a cytoplasmic antigen of equine mononuclear phagocytes.

An IgG1 mouse monoclonal antibody, designated 1.646, is described which recognizes a cytoplasmic antigen of equine mononuclear phagocytes. Indirect fluorescent antibody staining of peripheral blood leukocytes reveals a granular cytoplasmic staining, predominantly in adherent blood mononuclear cells. Indirect fluorescent antibody staining is positive for alveolar and peritoneal macrophages. In some horses, a few neutrophils are also stained. In equine tissue samples stained by immunohistochemistry, the distribution of positive cells is consistent with the distribution of tissue macrophages. The most intense and reliable staining occurs with splenic and lymph node macrophages. Hepatic Kupffer cells also stain with antibody 1.646, although the intensity of that staining is somewhat variable between horses. A granular pattern of staining typical of lipofuscin deposition is also seen in liver sections. There is also pale staining of some biliary and renal tubular epithelium. Equine erythrocytes, platelets and lymphocytes are not recognized by this antibody, and neither are monocyte/macrophages of human, canine or feline origin. Antibody 1.646 recognizes two proteins (150 and 30 kDa) of equine monocyte-derived macrophages when assayed by Western immunoblot. Because of the distribution of staining (tissue mononuclear phagocytes, lipofuscin-containing storage granules, biliary and renal tubular epithelium, and some neutrophils) we hypothesize that antibody 1.646 recognizes a cytoplasmic antigen that is closely associated with lysosomal membranes.

BPV-4 induces amplification and activation of 'silent' BPV-1 in a sub-line of C127 cells.

BPV-4-induced malignant transformation of C127 mouse fibroblasts in vitro is the result of a 'hit and run' mechanism. Viral DNA is lost on continued subculture of transformed cell lines without loss of any malignant characteristics. The DNA from these cells harbours specific amplified sequences. Two such amplified fragments of approximately 10 kb and 12 kb were molecularly cloned and designated HL-10 and HL-12 respectively. HL-10 transformed C127 cells efficiently and therefore encodes a transforming function, whereas the 12 kb clone did not. Heteroduplexes showed that HL-12 was homologous to HL-10 except for two additional tandem copies of an approximately 1.7 kb sequence. Sequence analysis of HL-10 revealed that the clone contained a 5.2 kb region from BPV-1 including the transforming ORFs. Transformation studies have shown differences between HL-10 and BPV-1, indicating that the host flanking sequences may contribute to the transforming potential of the BPV-1 ORFs. The BPV-1 DNA was associated with sequences homologous to murine autonomously replicating sequences (ARS) implicated in the establishment of multiple tandem DNA repeats. As the parental cells contain the set of sequences amplified in transformed cells in single copy and show none of the characteristics of transformed cells, we conclude that BPV-4 has activated these sequences by amplification and rearrangement. These phenomena may be mediated through an interaction between BPV-4 proteins and the BPV-1 origin of DNA replication or via the ARS region.

Intrahelical pseudoknots and interhelical associations mediated by mispaired human minisatellite DNA sequences in vitro.

The human minisatellite arrays, 33.6 and 33.15, consist of tandem reiterations of a 37-nucleotide (nt) and a 16-nt repeat unit sequence, respectively, both of which contain a majority of purine bases on one strand. Knot-like tertiary structures, which mapped to the cloned arrays, were observed by electron microscopy (EM) in homoduplex molecules produced by denaturation and reannealing in vitro. They result from a primary hybridization between misaligned repeat units of the array, forming a slipped-strand structure with staggered single-stranded DNA loops, followed by a secondary hybridization between repeat units in the two loops. Depending on the relative alignment of the loops when they hybridize, a particular form of intrahelical pseudoknot is produced. Theta-shaped, figure-of-eight, and bow-shaped structures were the most common conformational isomers observed in homoduplexes flattened into two dimensions during EM preparation. At the site of a bow-shaped structure, a conformation-dependent bend of approximately 60 degrees between the flanking DNA segments is induced; the other conformations generally do not deflect the line of the main DNA axis. Paired loops, similar to the bow-shaped structure, were apically situated in some supercoiled plasmids containing the 33.6 array. Both plasmids formed intermolecular associations, consisting of two (or more) homoduplex molecules held together at or immediately adjacent to a nexus which mapped to the minisatellite sequences. These associations might arise either by interhelical hybridization between arrays or by knot-like structures interfering with branch migration of chi-form Holliday junctions.

Wild-type equine infectious anemia virus replicates in vivo predominantly in tissue macrophages, not in peripheral blood monocytes.

In situ hybridization of tissues from two horses infected with the wild-type Wyoming strain of equine infectious anemia virus (EIAV) identified the liver, spleen, lymph nodes, kidney, lung, and adrenal gland as the primary host tissue sites for viral transcription during acute infection. Combined immunohistochemistry, with a monoclonal antibody recognizing a cytoplasmic antigen of equine mononuclear phagocytes, and in situ hybridization for viral RNA identified most infected cells as mature tissue macrophages. In contrast, in situ hybridization of adherent peripheral blood mononuclear cells collected from horses on various days during the first 2 weeks postinfection with the Wyoming strain of EIAV failed to detect any viral RNA in these cells. For the two horses described here, serum reverse transcriptase activity correlated directly with the degree of replication detected in tissue macrophages on the day of sacrifice. These results suggest that unlike other lentivirus infections in which mature tissue macrophages accumulate cytoplasmic viral RNA to a high level but fail to produce infectious virions, mature tissue macrophages are the likely primary source of the high titer of viremia present during acute infection with EIAV. No significant posttranscriptional block of viral replication in tissue macrophages appears to occur with EIAV.

The surface envelope protein gene region of equine infectious anemia virus is not an important determinant of tropism in vitro.

Virulent, wild-type equine infectious anemia virus (EIAV) is restricted in one or more early steps in replication in equine skin fibroblast cells compared with cell culture-adapted virus, which is fully competent for replication in this cell type. We compared the sequences of wild-type EIAV and a full-length infectious proviral clone of the cell culture-adapted EIAV and found that the genomes were relatively well conserved with the exception of the envelope gene region, which showed extensive sequence differences. We therefore constructed several wild-type and cell culture-adapted virus chimeras to examine the role of the envelope gene in replication in different cell types in vitro. Unlike wild-type virus, which is restricted by an early event(s) for replication in equine dermis cells, the wild-type outer envelope gene chimeras are replication competent in this cell type. We conclude that even though there are extensive sequence differences between wild-type and cell culture-adapted viruses in the surface envelope gene region, this domain is not a determinant of the differing in vitro cell tropisms.

Immune-mediated thrombocytopenia in horses infected with equine infectious anemia virus.

An adult horse infected with a virulent, cell culture-adapted strain of equine infectious anemia virus (EIAV) developed cyclical thrombocytopenia in which the nadir of platelet counts coincided with peak febrile responses. In order to investigate the mechanism of thrombocytopenia during acute febrile episodes, four adult horses were experimentally infected with the wild-type Wyoming strain of EIAV. Platelet counts decreased from baseline as rectal temperature increased. Serum reverse transcriptase activity increased above background levels in all horses, coincident with increase in rectal temperature. All horses developed an EIAV-specific immune response detectable by Western immunoblot by postinfection day 10. Increases in platelet-associated immunoglobulins G and M were detectable by direct fluorescent-antibody test and flow cytometric assay. Viral replication in bone marrow megakaryocytes was not detectable by in situ hybridization. Results suggest an immune-mediated mechanism of thrombocytopenia in horses infected with EIAV. Despite an inability to identify virion particles in association with platelet-bound antibody, the cyclical nature of the thrombocytopenia and the occurrence of a marked cell-free viremia concomitant with fever and thrombocytopenia suggest immune complex deposition on platelets. We propose that clearance of virus and antibody-coated platelets from the peripheral circulation by hepatic Kupffer cells and splenic macrophages may target infectious virus particles, in the form of immune complexes, to host cells most permissive for in vivo viral replication.

Equine infectious anemia virus derived from a molecular clone persistently infects horses.

A full-length molecular clone of equine infectious anemia virus (EIAV) was isolated from a persistently infected canine fetal thymus cell line (Cf2Th). Upon transfection of equine dermis cells, the clone, designated CL22, yielded infectious EIAV particles (CL22-V) that replicated in vitro in both Cf2Th cells and an equine dermis cell strain. Horses infected with CL22-V developed an antibody response to viral proteins and possessed viral DNA in peripheral blood mononuclear cells, as determined by polymerase chain reaction assays. In addition, horses infected with CL22-V became persistently infected and were capable of transmitting the infection by transfer of whole blood to uninfected horses. However, CL22-V, like the parental canine cell-adapted virus, did not cause clinical signs in infected horses. Reverse transcriptase assays of CL22-V- and virulent EIAV-infected equine mononuclear cell cultures indicated that the lack of virulence of CL22-V was not due to an inability to infect and replicate in equine mononuclear cells in vitro.

Distribution of equine infectious anemia in equids in southeastern United States.

State veterinarians in 11 southeastern states completed a questionnaire designed to determine the proportion of equids in the region that were seropositive for equine infectious anemia (EIA). Cases of EIA were diagnosed in each of the states surveyed. Distinct geographic clusters of cases were apparent in Tennessee and Kentucky adjacent to the Mississippi River, in the Piedmont of North Carolina at the Virginia border, in north central Georgia, and throughout the Florida peninsula. It is suggested that the national EIA program could be improved by standardization and wider application of uniform active surveillance measures, and improved documentation of EIA status of horses on acquisition and transfer records.

Characterisation of an extrachromosomal DNA element from Theileria annulata.

Extrachromosomal nucleic acid elements are found in all organisms, commonly as organelle, viral or plasmid genomes. In this paper we describe the initial characterisation of a novel 6.5-kb linear, double-stranded extrachromosomal element from Theileria annulata, and a 2.6-kb RNA species. The DNA element is present in different stages of the life cycle and in different stocks of the parasite. Northern blots of total RNA isolated from different stages of the parasite, probed with the purified element, detect three major transcripts, of 1.45, 1.05 and 0.24 kb, present in all life-cycle stages examined. The possible origin and function of this element is discussed, together with its possible use as a transfection vector for the introduction of genes into protozoan cells.

Multiple Harvey-ras genes in the bovine genome.

Three different Harvey-ras (Ha-ras) genes have been identified in the bovine genome by screening a lambda phage-bovine DNA library with the ras gene of the Harvey murine sarcoma virus. The genes have been characterized by hybridization and heteroduplex analysis with both the viral and human Ha-ras genes. Based on heteroduplex mapping, the putative direction of transcription and the approximate position of the coding regions of the bovine genes were established. Two genes, bovine Ha-ras 1 and 2, both show an arrangement of exons and introns typical of c-Ha-ras genes. However, they are distinct entities as they have different nucleotide sequences and physical maps, and heteroduplexes between them contain a region of non-homology upstream of exon 1. The third gene, c-Ha-ras 3, does not contain introns and is a pseudogene, analogous to human c-Ha-ras 2. The nucleotide sequence of both Ha-ras 1 and Ha-ras 2 has been determined and shows that Ha-ras 1 encodes a bona fide ras protein, whereas Ha-ras 2 has diverged considerably from a recognizably functional sequence.

DNA tertiary structures formed in vitro by misaligned hybridization of multiple tandem repeat sequences.

DNA tertiary structures are shown to be formed by denaturation and reannealing in vitro of molecularly-cloned DNA containing multiple tandem repeat sequences. Electron microscopy of homoduplex DNA molecules containing the human c-Harvey-ras gene revealed knot-like structures which mapped to the position of the 812 bp variable tandem repeat (VTR) sequence. We propose that the structures result from slipped-strand mispairing within the VTR and hybridisation of homologous repetitive sequences in the single-stranded loops so produced. Similar structures were also found in freshly-linearized supercoiled plasmids. More complex knot-like structures were found in homoduplexes of a 4 kb tandem array from the hypervariable region 3' to the human alpha-globin locus. Formation of such DNA tertiary structures in vitro also provides a practical method for identifying and mapping direct tandem repeat arrays that are at least 800 bp long.

R-loop mapping of RNA transcripts from bovine papillomavirus type 4.

RNA isolated from bovine oesophageal warts was hybridised to BPV-4 genomic DNA cloned in a plasmid vector. In R-loop preparations, six major classes of transcripts were mapped. Class I, due to an unspliced transcript encompassing the E4/E5 ORF region, is most common. In Class II the E4/E5 region is spliced at its 5' end to the E6 ORF region, and the RNAs appear to have different transcription start points in the E6 ORF. Some Class I R-loops may represent shorter Class II-type transcripts not hybridised to the E6 region. Transcripts that form Class III R-loops have not been previously described for BPV-4 and contain the E4/E5 ORF exon spliced at its 3' end to the LI ORF. In Class IV, transcripts map to the 3' end of the EI exon, artificially truncated by the Bam HI site used for cloning the BPV-4 genome, and are spliced to the 5' end of the exon containing the E4/E5 ORF. Class V transcripts are ambiguously located in the cloned BPV-4 genome, and could be derived from the EI or LI ORF. The former case may represent the remainder of the transcript from Class IV R-loops. The rare Class VI R-loops are due to the L2 ORF spliced at its 3' end to the LI ORF.

Rectal transmission of bovine leukemia virus in cattle and sheep.

Bovine leukemia virus (BLV) was transmitted by rectal inoculation of BLV-infective whole blood into cattle and sheep. Two cows and 2 sheep each were given 500 ml and 50 ml of blood, respectively, by rectal infusion. Two sheep which served as positive controls each were given 1 ml of the same blood, IV. All animals became seropositive to BLV by postinoculation week 5. Although relatively large volumes of blood were used for rectal inoculation, a base line for infectivity was established for the rectal route.

Gene amplification: the Chinese hamster glutamine synthetase gene.

The glutamine analogue, methionine sulphoximine, has been used to develop Chinese hamster ovary cell lines which overproduce the enzyme glutamine synthetase. Southern and Northern blot hybridization studies showed that this overproduction is due to gene amplification of a region of DNA containing the glutamine synthesis gene coding sequences, with a corresponding increase in glutamine synthetase mRNA and protein. This amplification has increased the glutamine synthetase gene copy number to approximately 1000 copies per cell. Genomic DNA sequences from the amplified region have been cloned and the structure of the glutamine synthetase gene within this region is being analysed.

Sequence homologies between bovine papillomavirus genomes mapped by a novel low-stringency heteroduplex method.

The bovine papillomaviruses (BPVs) types 1, 2, and 5 cause fibropapillomas whereas BPVs types 3, 4, and 6 cause true papillomas. A novel method of heteroduplex mapping at low stringency of hybridisation has identified the position and relative orientation of distantly related sequences in the genomes of these viruses. The genomes of BPV-1 and BPV-2 are closely related but both show a high degree of sequence divergence from the BPV-5 genome. A 1.25-kb sequence adjacent to the unique BamHI site of the BPV-5 genome hybridised to BPV-1 and to the equivalent region of BPV-2. The hybridising sequence in the BPV-1 genome mapped to the C-terminal region of the E1 open reading frame (ORF) and the N-terminal region of the E2 ORF. The BPV-3, BPV-4, and BPV-6 genomes show moderate homology to each other but minimal homology to the fibropapillomavirus genomes. Low-stringency heteroduplex mapping revealed that overlapping sequences in the BPV-1 E1 and L1 ORFs (or the equivalent regions in BPV-2) hybridised to sequences in BPV-3, BPV-4, and BPV-6. Hybrid regions were less than 1 kb long and were sometimes interrupted by short nonhybridising segments. The hybridising sequences in BPV-3 and BPV-4 are positioned in a way that parallels the spacing of the E1 and L1 ORFs in BPV-1. These data suggest that the bovine fibropapilloma viruses and true papilloma viruses share a similar genomic organization, but have undergone extensive sequence divergence.

Equine herpesvirus type 2: cell-virus relationship during persistent cell-associated viremia.

Some aspects of the biology of equine herpesvirus type 2 (EHV-2) were investigated by examination of the persistent cell-associated viremia stage of the infection. The EHV-2 infection of leukocytes was latent, because free virus was not retrieved without first cultivating harvested leukocytes in vitro. A virus infective center (IC) assay was developed to enumerate latently infected cells in the leukocyte population. This assay proved to be simple and reproducible and revealed a linear relationship between IC plaques formed and the number of cells inoculated, except where large numbers of cells (greater than 4 X 10(6)) were inoculated per 10 cm2 dish. This reduction at high cell densities of IC/10(6) cells inoculated was dependent on cells obtained from an EHV-2-infected horse. There was considerable variation in the numbers of IC/10(6) leukocytes harvested from different horses, but little variation in the harvests from the same horse at different times. There seemed to be a direct relationship between serum-neutralization titers and IC numbers. Transfer of viable infected leukocytes to 2 fetuses failed to establish EHV-2 infection. Infection of equine fetal kidney cells with EHV-2 virus failed to produce detectable Fc receptors on the cell surface.