David Onions

The Fecal Viral Flora of California Sea Lions

ABSTRACT California sea lions are one of the major marine mammal species along the Pacific coast of North America. Sea lions are susceptible to a wide variety of viruses, some of which can be transmitted to or from terrestrial mammals. Using an unbiased viral metagenomic approach, we surveyed the fecal virome in California sea lions of different ages and health statuses. Averages of 1.6 and 2.5 distinct mammalian viral species were shed by pups and juvenile sea lions, respectively. Previously undescribed mammalian viruses from four RNA virus families (Astroviridae, Picornaviridae, Caliciviridae, and Reoviridae) and one DNA virus family (Parvoviridae) were characterized. The first complete or partial genomes of sapeloviruses, sapoviruses, noroviruses, and bocavirus in marine mammals are reported. Astroviruses and bocaviruses showed the highest prevalence and abundance in California sea lion feces. The diversity of bacteriophages was higher in unweaned sea lion pups than in juveniles and animals in rehabilitation, where the phage community consisted largely of phages related to the family Microviridae. This study increases our understanding of the viral diversity in marine mammals, highlights the high rate of enteric viral infections in these highly social carnivores, and may be used as a baseline viral survey for comparison with samples from California sea lions during unexplained disease outbreaks.

Identification of Exogenous Forms of Human-Tropic Porcine Endogenous Retrovirus in Miniature Swine

ABSTRACT The replication of porcine endogenous retrovirus subgroup A (PERV-A) and PERV-B in certain human cell lines indicates that PERV may pose an infectious risk in clinical xenotransplantation. We have previously reported that human-tropic PERVs isolated from infected human cells following cocultivation with miniature swine peripheral blood mononuclear cells (PBMC) are recombinants of PERV-A with PERV-C. Here, we report that these recombinants are exogenous viruses in miniature swine; i.e., they are not present in the germ line DNA. These viruses were invariably present in miniature swine that transmitted PERV to human cells and were also identified in some miniature swine that lacked this ability. These data, together with the demonstration of the absence of both replication-competent PERV-A and recombinant PERV-A/C loci in the genome of miniature swine (L. Scobie, S. Taylor, J. C. Wood, K. M. Suling, G. Quinn, C. Patience, H.-J. Schuurman, and D. E. Onions, J. Virol. 78:2502-2509, 2004), indicate that exogenous PERV is the principal source of human-tropic virus in these animals. Interestingly, strong expression of PERV-C in PBMC correlated with an ability of the PBMC to transmit PERV-A/C recombinants in vitro, indicating that PERV-C may be an important factor affecting the production of human-tropic PERV. In light of these observations, the safety of clinical xenotransplantation from miniature swine will be most enhanced by the utilization of source animals that do not transmit PERV to either human or porcine cells. Such animals were identified within the miniature swine herd and may further enhance the safety of clinical xenotransplantation.

Detection of Canine Herpesvirus 1 in a wide range of tissues using the polymerase chain reaction

Canine herpesvirus 1 (CHV-1), a member of the alphaherpesvirus sub-family, is known to cause fatal infections in litters of puppies and may also be involved in infertility, abortion, and stillbirths in adult dogs. The purpose of this study was to determine the presence of CHV-1 DNA using the polymerase chain reaction (PCR) in twelve key sites that have been associated with latency for the other herpesviruses. A 605 base pair portion of the viral glycoprotein B (gB) gene was amplified using degenerate primers, cloned, and sequenced. Conventional 20 mer primers were designed using this sequence information to amplify a 120 bp fragment of gB situated between the original degenerate primers. The specificity of amplification was confirmed by Southern Blot hybridisation using an internal oligonucleotide probe. DNA was extracted from tissue samples taken from twelve dogs at post mortem and from twenty-four blood samples. Nine out of twelve dogs showed evidence of infection with CHV-1; the tissues most commonly affected were lumbo-sacral ganglia (5/12 dogs), tonsil (5/12), parotid salivary gland (4/9), and liver (4/9). No positive results were detected within the twenty-four blood samples. These results indicate that exposure to CHV-1 may be much more common than previously suggested.

Feline leukemia virus DNA vaccine efficacy is enhanced by coadministration with interleukin-12 (IL-12) and IL-18 expression vectors

ABSTRACT The expectation that cell-mediated immunity is important in the control of feline leukemia virus (FeLV) infection led us to test a DNA vaccine administered alone or with cytokines that favored the development of a Th1 immune response. The vaccine consisted of two plasmids, one expressing the gag/pol genes and the other expressing the env gene of FeLV-A/Glasgow-1. The genetic adjuvants were plasmids encoding the feline cytokines interleukin-12 (IL-12), IL-18, or gamma interferon (IFN-γ). Kittens were immunized by three intramuscular inoculations of the FeLV DNA vaccine alone or in combination with plasmids expressing IFN-γ, IL-12, or both IL-12 and IL-18. Control kittens were inoculated with empty plasmid. Following immunization, anti-FeLV antibodies were not detected in any kitten. Three weeks after the final immunization, the kittens were challenged by the intraperitoneal inoculation of FeLV-A/Glasgow-1 and were then monitored for a further 15 weeks for the presence of virus in plasma and, at the end of the trial, for latent virus in bone marrow. The vaccine consisting of FeLV DNA with the IL-12 and IL-18 genes conferred significant immunity, protecting completely against transient and persistent viremia, and in five of six kittens protecting against latent infection. None of the other vaccines provided significant protection.

Absence of Replication-Competent Human-Tropic Porcine Endogenous Retroviruses in the Germ Line DNA of Inbred Miniature Swine

ABSTRACT The potential transmission of porcine endogenous retroviruses (PERVs) has raised concern in the development of porcine xenotransplantation products. Our previous studies have resulted in the identification of animals within a research herd of inbred miniature swine that lack the capacity to transmit PERV to human cells in vitro. In contrast, other animals were capable of PERV transmission. The PERVs that were transmitted to human cells are recombinants between PERV-A and PERV-C in the post-VRA region of the envelope (B. A. Oldmixon, J. C. Wood, T. A. Ericsson, C. A. Wilson, M. E. White-Scharf, G. Andersson, J. L. Greenstein, H. J. Schuurman, and C. Patience, J. Virol. 76:3045-3048, 2002); these viruses we term PERV-A/C. This observation prompted us to determine whether these human-tropic replication-competent (HTRC) PERV-A/C recombinants were present in the genomic DNA of these miniature swine. Genomic DNA libraries were generated from one miniature swine that transmitted HTRC PERV as well as from one miniature swine that did not transmit HTRC PERV. HTRC PERV-A/C proviruses were not identified in the germ line DNAs of these pigs by using genomic mapping. Similarly, although PERV-A loci were identified in both libraries that possessed long env open reading frames, the Env proteins encoded by these loci were nonfunctional according to pseudotype assays. In the absence of a germ line source for HTRC PERV, further studies are warranted to assess the mechanisms by which HTRC PERV can be generated. Once identified, it may prove possible to generate animals with further reduced potential to produce HTRC PERV.

Tumours derived from HTLV-I tax transgenic mice are characterized by enhanced levels of apoptosis and oncogene expression

In order to investigate the role that the human T‐lymphotropic virus type I (HTLV‐I) tax oncogene plays in apoptosis and transformation in vivo, four lines of HTLV‐I tax transgenic mice were generated under the regulatory control of the CD3‐ϵ promoter–enhancer sequence. These mice develop a variety of phenotypes including mesenchymal tumours, which develop at wound sites, and salivary and mammary adenomas. In situ DNA fragment labelling and immunocytochemical analysis of these tumours reveals that they display enhanced levels of apoptosis, which is associated with elevated levels of Myc, Fos, Jun, and p53 protein expression. Furthermore, double immunofluorescent staining shows that Tax expression and apoptosis co‐localize, indicating that Tax expression is closely associated with apoptosis in vivo. Copyright

Complete DNA sequence of canine adenovirus type 1.

The complete DNA sequence of a field strain of canine adenovirus type 1 was determined by sequencing random fragments of viral DNA cloned into pBluescript. The virus has a genome of 30536 bp flanked by two identical 161 bp inverted terminal repeats. Thirty ORFs have been identified, based on genomic location or sequence identity with published adenoviruses. These are arranged into similar discrete regions found in the human adenoviruses. ORFs in the late region show greatest identity with published human adenovirus sequences, whereas the E3 and E4 ORFs show little or none.

Validation of the safety of MDCK cells as a substrate for the production of a cell-derived influenza vaccine.

Abstract Cell culture-based production methods may assist in meeting increasing demand for seasonal influenza vaccines and developing production flexibility required for addressing influenza pandemics. MDCK-33016PF cells are used in propagation of a cell-based seasonal influenza vaccine (Optaflu®); but, like most continuous cell lines, can grow in immunocompromised mice to produce tumors. It is, therefore, essential that no residual cells remain within the vaccine, that cell lysates or DNA are not oncogenic, and that the cell substrate does not contain oncogenic viruses or oncogenic DNA. Multiple, redundant processes ensure the safety of influenza vaccines produced in MDCK-33016PF cells. The probability of a residual cell being present in a dose of vaccine is approximately 1 in 1034. Residual MDCK-DNA is ≤10ng per dose and the ß-propiolactone used to inactivate influenza virus results in reduction of detectable DNA to less than 200base pairs (bp). Degenerate PCR and specific PCR confirm exclusion of oncogenic viruses. The manufacturing process has been validated for its capacity to remove and inactivate viruses. We conclude that the theoretical risks arising from manufacturing seasonal influenza vaccine using MDCK-33016PF cells are reduced to levels that are effectively zero by the multiple, orthogonal processes used during production.

Haemopoietic colony formation (BFU-E, GM-CFC) during the development of pure red cell hypoplasia induced in the cat by feline leukaemia virus

The GM-CFC assay for granulocyte-macrophage progenitors and the BFU-E and CFU-E assay for early and late erythroid progenitors from cat bone marrow were characterized. GM-CFC gave 59 +/- 4 to 118 +/- 6 colonies per 10(5) bone marrow cells using colony stimulating factors (CSF) from cat, mouse or human sources. The CFU-E and BFU-E assays gave 114 +/- 7 and 58 +/- 7 colonies respectively with optimum doses of erythropoietin. Irradiated cat bone marrow cells were good sources of CSF and of burst promoting activity for these assays. Kittens infected with feline leukaemia virus, subgroup C (FeLV-C), which induces pure red cell hypoplasia, showed the incidence of BFU-E decreased to 25-35% of controls as early as one week postinfection, and even lower values at later times. In contrast, the incidence of GM-CFC remained normal for several weeks. No evidence of inhibitory cells or of lack of stimulatory cells in the infected marrows was seen when they were cultured together with normal marrow in the BFU-E assay. Conversely, normal marrow cells were not able to restore BFU-E growth from infected marrow. This suggests a direct action of FeLV-C on early erythroid precursors. Infection with FeLV, subgroup A, which induces only a mild transitory anaemia, produces only a moderate decrease in the incidence of BFU-E.

Effect of hydrocortisone on osteoclasts generated in cat bone marrow cultures.

SummaryThe generation of osteoclasts in cultures of cat bone marrow was completely inhibited for 4 weeks with 10−6M hydrocortisone (HC) and partially inhibited with 10−7 to 10−9M in a dose-dependent fashion. This effect was completely reversible when cultures were exposed for only 2 weeks to 10−9 or 10−8M HC. However, cultures in which higher concentrations (10−7 to 10−5M) were maintained for the same period did not show complete recovery in terms of numbers of osteoclasts and number of nuclei per cell after withdrawal of HC, suggesting that precursor cells of osteoclasts were also damaged by HC. To study the effects of HC on osteoclasts already present in the cultures, 10−6M was added to 4-week-old untreated cultures. The number of osteoclasts decreased rapidly and a gross morphological response was also apparent (rounding of the cells leading to detachment from the substratum and inhibition of cell fusion), indicating that the generation as well as the survival of osteoclasts in vitro are sensitive to HC. The morphological changes observed under optical and electron microscopy correspond to those of the reported inactive form of osteoclasts, and suggest that their function may also be altered by HC.