Fibroma Virus Research Articles

Comparative characterization of Leporipoxvirus members’ reproduction in continuous cell culture

Examination of the virus-cell interactions is of both scientific and practical importance. Our study was aimed at comparative characterization of rabbit myxoma virus and Shope fibroma virus biological properties that manifested during the virus reproduction in RK-13/2-03 clonal continuous rabbit kidney cell culture. It was demonstrated that the viruses varied in infection development and cytopathic effect duration in RK-13/2-03 cell culture. Apparent lesions in cell monolayers infected by myxoma virus and fibroma virus at similar multiplicity of infection and cultivation temperature were observed on day 2 and day 3 of cultivation, respec- tively, as well as maximum cell lesions with evident degeneration were observed on day 3 and day 6 of cultivation, respectively. Myxoma virus was accumulated at titre of 6.25–6.50 lg TCID 50 /0,2 cm 3 , and Shope fibroma virusа was accumulated at titre of 5.50−5.75 lg TCID 50 /0.2 cm 3 . Shope fibroma virus demonstrated such infectivity during three passages and myxoma virus demonstrated such infectivity during twenty passages. Prepared cultures were identified as myxoma virus and Shope fibroma virus with molecular genetic analysis. Tests of the viruses for their antigenic relatedness showed that antibodies against myxoma virus were able to neutralize Shope fibroma virus also. NT titres of antibodies against both viruses were similar (1:8). RK-13/2-03 cell culture was found to be highly permissive to Shope fibroma virus that had been isolated from the diseased rabbit and not been an attenuated variant.

MORBIDITY AND MORTALITY IN ONTARIO RODENTS AND LAGOMORPHS: A 30-YEAR RETROSPECTIVE REVIEW.

Passive surveillance is an important component of wildlife health surveillance that allows for the identification of emerging pathogens as well as population-level threats. We investigated the most common causes of morbidity and mortality in rodents and lagomorphs submitted to the Canadian Wildlife Health Cooperative (CWHC) in Ontario and the Ontario Veterinary College (OVC) over a 30-yr period. A total of 836 cases representing 13 species of rodents and three species of lagomorph were submitted to the CWHC and the OVC wildlife pathology service. Infectious or inflammatory diagnoses were most common in our data set, followed by trauma and unknown diagnoses. The most frequently identified primary diagnosis was encephalitis with histological lesions consistent with neural larva migrans including the presence of inflammation and malacia of brain tissue and, in some cases, characteristic nematode larvae. Other infectious diagnoses were squirrel fibroma virus and Toxoplasma gondii infections. Knowledge of common pathogens observed in various species of rodents and lagomorphs can aid in triage and treatment decisions at veterinary clinics and wildlife rehabilitation centers, and guide sample collection and test requisition at post-mortem examination.

Generation of recombinant MVA-norovirus: a comparison study of bacterial artificial chromosome- and marker-based systems

BackgroundRecombinant Modified Vaccinia Virus Ankara has been employed as a safe and potent viral vector vaccine against infectious diseases and cancer. We generated recMVAs encoding norovirus GII.4 genotype capsid protein by using a marker-based approach and a BAC-based system. In the marker-based approach, the capsid gene together with a reporter gene was introduced into the MVA genome in DF-1 cells. Several rounds of plaque purification were carried out to get rid of the WT-MVA. In the BAC-based approach, recMVA-BAC was produced by en passant recombineering in E. coli. Subsequently, the recMVAs were rescued in DF-1 cells using a helper rabbit fibroma virus. The BAC backbone and the helper virus were eliminated by passaging in DF-1 cells. Biochemical characteristics of the recMVAs were studied.ResultsWe found the purification of the rare spontaneous recombinants time-consuming in the marker-based system. In contrast, the BAC-based system rapidly inserted the gene of interest in E. coli by en passant recombineering before virion production in DF-1 cells. The elimination of the reporter gene was found to be faster and more efficient in the BAC-based approach. With Western blotting and electron microscopy, we could prove successful capsid protein expression and proper virus-assembly, respectively. The MVA-BAC produced higher recombinant virus titers and infected DF-1 cells more efficiently.ConclusionsComparing both methods, we conclude that, in contrast to the tedious and time-consuming traditional method, the MVA-BAC system allows us to quickly generate high titer recMVAs.

Construction of an infectious horsepox virus vaccine from chemically synthesized DNA fragments.

Edward Jenner and his contemporaries believed that his variolae vaccinae originated in horses and molecular analyses show that modern vaccinia virus (VACV) strains share common ancestry with horsepox virus (HPXV). Given concerns relating to the toxicity of modern VACV vaccines, we asked whether an HPXV-based vaccine might provide a superior alternative. Since HPXV may be extinct and the only specimen of HPXV that has been identified is unavailable for investigation, we explored whether HPXV could be obtained by large-scale gene synthesis. Ten large (10–30 kb) fragments of DNA were synthesized based on the HPXV sequence along with two 157 nt VACV terminal sequences, and were recombined into a live synthetic chimeric HPXV (scHPXV) in cells infected with Shope fibroma virus (SFV). Sequencing of the 212 kbp scHPXV confirmed it encoded a faithful copy of the input DNA. We believe this is the first complete synthesis of a poxvirus using synthetic biology approaches. This scHPXV produced smaller plaques, produced less extracellular virus and exhibited less virulence in mice than VACV, but still provided vaccine protection against a lethal VACV challenge. Collectively, these findings support further development of scHPXV as a novel replication-proficient smallpox vaccine.

Pathology in practice. Cutaneous and disseminated infection with squirrel fibroma virus (SFV).

Pathology in practice. Cutaneous and disseminated infection with squirrel fibroma virus (SFV).

Evolution of viral sensing RIG-I-like receptor genes in Leporidae genera Oryctolagus, Sylvilagus, and Lepus

One of the most severe European rabbit (Oryctolagus cuniculus) pathogens is myxoma virus (MYXV), a rabbit-specific leporipoxvirus that causes the highly lethal disease myxomatosis. Other leporid genera, Sylvilagus and Lepus, encompass species with variable susceptibilities to MYXV, but these do not develop the lethal form of the disease. The protective role of the retinoic acid-inducible gene-I (RIG-I/DDX58) in sensing MYXV in nonpermissive human myeloid cells prompted the study of the RIG-I-like receptor (RLR) family evolution in the three leporid genera. This viral-sensor family also includes the melanoma differentiation-associated factor 5 (MDA5/IFIH1), and the laboratory of genetics and physiology 2 (LGP2/DHX58). Considering specifically the MYXV susceptible host (European rabbit) and one of the virus natural long-term hosts (Sylvilagus bachmani, brush rabbit), the amino acid differences of positively selected sites in RIG-I between the two species were located in the protein region responsible for viral RNA recognition and binding, the repressor domain. Such differences might play a determinant role in how MYXV is sensed. When looking for episodic selection on MDA5 and LGP2 of the eastern cottontail (Sylvilagus floridanus), we also uncovered evidence of selective pressures that might be exerted by a species-specific leporipoxvirus, the Shope fibroma virus. Finally, a putative alternative splicing case was identified in Oryctolagus and Lepus MDA5 isoforms, corresponding to the deletion of one specific exon. This study provided the first insights into the evolution of the leporid RLR gene family that helps illuminate the origins of the species-specific innate responses to pathogens and more specifically to MYXV.

Characterization of a Novel Poxvirus in a North American Red Squirrel (Tamiasciurus hudsonicus)

In 2009, a novel poxvirus was identified in a North American red squirrel (Tamiasciurus hudsonicus) from Yukon, Canada. Initial molecular analyses indicated that this virus was likely to be distinct from all other known mammalian poxviruses, including those previously associated with disease in tree squirrels--squirrel fibroma virus in North America and squirrelpox virus in the UK (UK SQPV). We characterize the Canadian squirrelpox virus (Canadian SQPV) using DNA sequence analysis and negative-contrast transmission electron microscopy (TEM). Sequence analysis revealed that the Canadian SQPV is distinct from all known mammalian poxviruses but most closely related to the parapoxviruses, followed by UK SQPV. In contrast, TEM showed that the ultrastructure of Canadian SQPV is distinct from that of the parapoxviruses and UK SQPV but indistinguishable from that of other chordopoxviruses (a morphological group that includes the orthopoxviruses and leporipoxviruses). Overall, our analyses suggest a potential evolutionary relationship between UK SQPV and Canadian SQPV and supports our assertion that the Canadian virus represents a newly identified poxvirus in North American tree squirrels.

Development and validation of aMyxoma virusreal-time polymerase chain reaction assay

To aid in the rapid diagnosis of myxomatosis in rabbits, a real-time polymerase chain reaction (PCR) for the specific detection of Myxoma virus is described. Primers and probe were designed to amplify a 147-bp fragment within the Serp2 gene. The assay was able to detect 23 copies of a synthesized oligo indicating a reliable sensitivity. In addition, the real-time PCR did not detect the Rabbit fibroma virus used in myxomatosis vaccines. The novel PCR was shown to be able to detect Myxoma virus in fresh and paraffin-embedded rabbit tissues originating from myxomatosis cases from various regions in Switzerland.

SEROLOGICAL RESPONSE TO MYXOMATOSIS VACCINATION BY DIFFERENT INOCULATION SYSTEMS ON FARM RABBITS.

In order to assess the serological response to different vaccination systems against myxomatosis, five groups of 20 rabbits each were inoculated with a vaccine prepared from the Shope fibroma virus (Mixohipra-FSA®). Administration systems differed between groups: subcutaneous injection (group A), intradermic inoculation in one-shot one-impact (group B), one-shot three- impacts (group C), two-shots one-impact (group D) and two shots three-impacts (group E). Clinical signs, general and local, as well as serological response at 28 days post-vaccination were compared among the different groups. Vaccination did not affect health status of rabbits but it produced nodules at the inoculation site, mainly in groups vaccinated by intradermic route where the presence and size of the nodules were closely related to serological response. Although no differences due to the administration system were found in the serological response (P>0.05), subcutaneous injection and two-shot intradermic administration provided the most homogeneous responses.

Cottontail Rabbit Papillomavirus in Langerhans Cells in Sylvilagus spp.

A wildlife sanctuary presented an adult female cottontail rabbit (Sylvilagus spp.), age unknown, to the Colorado State University Pathology service for postmortem examination. Gross examination revealed numerous pigmented wartlike lesions arising from the skin of the head surrounding the ears, eyes, nares, mouth, and dorsum. Masses were firm, friable, and easily detached from the underlying skin. Differential diagnoses included Cottontail rabbit papillomavirus, Rabbit fibroma virus, and Myxoma virus. Histological examination revealed multiple papillary masses lined by stratified squamous epithelial cells with central cores of fibrovascular connective tissue and parakeratotic hyperkeratosis. Cells of the Stratum spinosum were frequently swollen with abundant perinuclear, cytoplasmic, clearing, and occasional intranuclear basophilic, glassy, spherical inclusions up to 3 microm in diameter. The lesions were consistent with Cottontail rabbit papillomavirus infection. Papilloma virus antigens were identified by immunohistochemistry. In addition, papillomavirus particles were identified by transmission electron microscopy within Langerhans cells of the epidermis, suggesting a unique mechanism for systemic dissemination of the virus. The present case report highlights the finding of viral particles within the Langerhans cells and suggests a novel mechanism of pathogenesis.

Poxvirus Infection in an American Red Squirrel (Tamiasciurus hudsonicus) from Northwestern Canada

There are two recognized poxviruses that are associated with disease in tree squirrels: squirrel fibroma virus (SQFV), Leporipoxvirus, which affects eastern grey squirrels (Sciurus carolinensis) in eastern North America, and squirrelpox virus (SQPV), a member of a newly identified poxvirus genus, which affects European red squirrels (Sciurus vulgaris) in the United Kingdom. In August 2008, a cutaneous poxvirus-associated disease was identified in a North American red squirrel (Tamiasciurus hudsonicus) from the Yukon Territory, Canada. The gross and microscopic appearance of the skin lesions was more consistent with SQPV than SQFV, and electron microscopy revealed poxvirions only within epithelial cells. Polymerase chain reaction (PCR) was used to identify poxvirus core protein encoding DNA in skin samples, and phylogenetic analysis showed that the inferred amino acid sequence was distinct from all other poxvirus species for which the core protein gene has been sequenced, including those of the genus Leporipoxvirus. Although the core protein sequence of SQPV was not available, comparison of the constructed phylogenetic tree to other published trees, based on major outer envelope proteins, revealed that the identified sequence occupies a position similar to SQPV in terms of its relationship to other poxviruses. However, PCR primers designed to amplify gene sequences encoding the SQPV major envelope protein and RNA polymerase did not amplify any sequences from infected tissues. These findings suggest that the virus present in this squirrel is a novel poxvirus of North American red squirrels. To our knowledge, this is the first case of poxvirus infection in Canadian squirrels outside of Ontario.

Cutaneous and Systemic Poxviral Disease in Red (Tamiasciurus hudsonicus) and Gray (Sciurus carolinensis) Squirrels

From September 2005 through October 2006, fibromatosis was diagnosed in 2 red squirrels (Tamiasciurus hudsonicus) and 1 gray squirrel (Sciurus carolinensis). All 3 squirrels had multifocal to coalescing, tan, firm alopecic cutaneous nodules. Two squirrels also had pulmonary nodules. Histologically, the cutaneous nodules had marked epidermal hyperplasia, with ballooning degeneration of keratinocytes, spongiosis, and eosinophilic cytoplasmic inclusions. The dermis was expanded by proliferation of atypical mesenchymal cells with cytoplasmic inclusions. Additional findings included pulmonary adenomatous hyperplasia with cytoplasmic inclusions, renal tubular epithelial hyperplasia with cytoplasmic inclusions, atypical mesenchymal proliferation in the liver, and atypical mesenchymal proliferation with cytoplasmic inclusions in the seminal vesicles. Ultrastructurally, poxviral particles were observed in skin scrapings and sections of cutaneous and pulmonary nodules. Polymerase chain reaction targeting the highly conserved Leporipoxvirus DNA polymerase gene was positive using DNA extracted from the cutaneous lesions of all 3 squirrels. Nucleotide sequence of the 390 base PCR amplicons was closely related to that of other members of the genus Leporipoxvirus. To the authors' knowledge, this is the first report of cutaneous and systemic poxviral disease in American red squirrels with molecular characterization of the squirrel fibroma virus.

A Shope Fibroma virus PYRIN-only protein modulates the host immune response.

PYRIN domain (PYD) proteins have recently emerged as important signaling molecules involved in the development of innate immunity to intracellular pathogens through activation of inflammatory mediator pathways. ASC is the central adaptor protein, which links pathogen recognition by PYD-containing pathogen recognition receptors to the activation of downstream effectors, including activation of Caspase-1 and NF-kappaB. The cellular PYD-only protein 1 (cPOP1) can block the recruitment of ASC to activated PAN receptors and thereby functions as an endogenous inhibitor of the PYD-mediated signal transduction pathway. Here we describe the identification and characterization of a Shope Fibroma homolog to cPOP1. Like cPOP1, a Shope Fibroma virus-encoded POP (vPOP), co-localizes and directly associates with ASC and inhibits PYD-mediated signal transduction. Poxviruses are known to encode immune evasive proteins to promote host cell infection and suppression of the host immune response. Poxvirus-encoded vPOPs represent a novel class of immune evasive proteins and impair the host response by blocking Cryopyrin and ASC inflammasome-mediated activation of pro-Caspase-1 and subsequent processing of pro-interleukin (IL)-1beta, and expression of vPOPs causes activation of NF-kappaB.

Shope fibroma virus keratitis and spontaneous cataracts in a domestic rabbit

A 7-year-old domestic rabbit presented for an enlarging ventral perilimbal mass OS. Keratectomy was performed to remove the mass. A diagnosis of Shope fibroma virus keratitis was confirmed based on signalment, clinical signs, histologic evaluation and virus isolation. Progression of bilateral cataracts leading to visual deficits was addressed with phacoemulsification. The rabbit remained visual and comfortable 5 months postoperatively and free of recurrence of the limbal mass 9 months after initial presentation.

A Shope Fibroma virus PYRIN-only protein modulates the host immune response

Classification of hepatitis C virus is based on phylogenetic analysis of the strains reported world wide. Different strains are classified within 6 major genotypes and several minor groups (subtypes). In addition to epidemiologic value of determining genotype/subtype of this virus, the result may change the therapeutic strategy used for a patient. During a survey on hepatitis C in Iran, we found two cases assigned as 1b genotype by PCR-RFLP on 5' UTR, but three based on core region sequencing. Fragments from 5' UTR, Core and NS5b regions were PCR-amplified and sequenced followed by phylogenetic analysis. Although the 5' UTR of this new strain is very similar to genotypes 1 and 6, analysis of core region classifies it in a separate branch of genotype 3, close to subtypes h and k. Further analysis of NS5b region put this new strain in a separate branch near other subtypes of genotype 3 and 4. These data are suggestive of a new subtype within genotype 3.

Variation in Ligand Binding Specificities of a Novel Class of Poxvirus-encoded Tumor Necrosis Factor-binding Protein

The Yatapoxviruses encode a distinct class of secreted TNF-binding protein (TNF-BP) that resembles an MHC class I heavy chain but distinct from any other known TNF inhibitor. Characterization of these viral TNF inhibitors from Tanapox virus, Yaba monkey tumor virus (YMTV) and a closely related version from Swinepox virus revealed dramatically differential TNF binding specificities for different mammalian species. The Tanapox virus 2L protein (TPV-2L) formed inhibitory complexes with human TNF, and interacted with monkey and canine TNF with high affinity but rabbit TNF with low affinity. On the other hand, YMTV-2L bound human and monkey TNF with high affinity but rabbit TNF with only low affinity. The TNF-BP from swinepox virus (SPV003/148) only interacted with porcine TNF with high affinity. The observed TNF binding analysis mirrored the biological activity of these TNF-binding protein to block TNF-induced cellular cytolysis. TPV-2L and YMTV-2L also inhibited the human TNF-mediated signaling in cells but TPV-2L exhibited higher affinity for human TNF (KD, 43 pm) compared with monkey (KD, 120 pm) whereas for YMTV-2L, the affinities were reversed (human TNF KD, 440 pm; monkey TNF KD, 230 pm). The interaction domain of human TNF with TNF-binding proteins is significantly different from that of TNFRs, as determined using human TNF mutants. We conclude that these poxvirus TNF-binding proteins represent a new class of TNF inhibitors and are distinct from the viral TNF receptor homologues characterized to date.

Tumorigenic Poxviruses Up-Regulate Intracellular Superoxide To Inhibit Apoptosis and Promote Cell Proliferation

Tumorigenic leporipoxviruses encode catalytically inactive homologs of cellular Cu-Zn superoxide dismutase (SOD1). The function of the orthologous myxoma virus M131R and Shope fibroma virus S131R gene products is uncertain, but they inhibit SOD1 activity by a process linked to binding its copper chaperone. Using a superoxide-sensitive dye (hydroethidine), we observed that virus infection increased intracellular superoxide levels in an M/S131R-dependent manner. To see whether this effect promotes infection, we deleted the Shope fibroma virus S131R gene and compared the clinical manifestations of wild-type and mutant virus infections in rabbits. S131RDelta virus produced significantly smaller fibroxanthosarcoma-like growths in vivo and, at a point where these growths were already receding, wild-type infections still showed extensive leukocyte infiltration, necrosis, and fibromatous cell proliferation. Coincidentally, whereas Jurkat cells are protected from mitochondria- and Fas-mediated apoptosis by wild-type myxoma virus in vitro, M131RDelta virus could not block Fas-initiated apoptosis as judged by DNA laddering, terminal deoxynucleotidyltransferase-mediated dUTP-fluorescein nick end labeling, and caspase 3 cleavage assays. These data suggest that tumorigenic poxviruses can modulate intracellular redox status to their advantage to stimulate infected cell growth and inhibit programmed cell death.

Targeted modification of the complete chicken lysozyme gene by poxvirus-mediated recombination

We have developed a novel ex vivo system for the rapid one-step targeted modification of large eucaryotic DNA sequences. The highly recombinant environment resulting from infection of rabbit cornea cells with the Shope fibroma virus was exploited to mediate precise modifications of the complete chicken lysozyme gene domain (21.5 kb). Homologous recombination was designed to occur between target DNA (containing the complete lysozyme gene domain) maintained in a lambda bacteriophage vector and modified targeting DNA maintained in a plasmid. The targeting plasmids were designed to transfer exogenous sequences (for example, beta-galactosidase alpha-complement, green fluorescent protein, and hydrophobic tail coding sequences) to specific sites within the lysozyme gene domain. Cotransfection of the target phage and a targeting plasmid into Shope fibroma virus infected cells resulted in the poxvirus-mediated transfer of the modified sequences from plasmid to phage. Phage DNA (recombinant and nonrecombinant) was then harvested from the total cellular DNA by packaging into lambda phage particles and correct recombinants were identified. Four different gene-targeting pairings were carried out, and from 3% to 11% of the recovered phages were recombinant. Using this poxvirus-mediated targeting system, four different regions of the chicken lysozyme gene domain have been modified precisely by our research group overall with a variety of inserts (6-971 bp), deletions (584-3000 bp), and replacements. We have never failed to obtain the desired recombinant. Poxvirus-mediated recombination thus constitutes a routine, rapid, and remarkably efficient genetic engineering system for the precise modification of large eucaryotic gene domains when compared with traditional practices.

Tumorigenic poxviruses: growth factors in a viral context?

Shope fibroma virus (SFV) is one of the few poxviruses that induce cutaneous tumours, whereas myxoma virus, a closely related leporipoxvirus, does not. However, both have a virally encoded homologue of the epidermal growth factor (namely SFGF and MGF, respectively) that is considered to be crucial for poxvirus tumorigenesis. In this study, the role of viral growth factors in the context of infection with SFV, a tumorigenic leporipoxvirus, was investigated. An SFV mutant was engineered with the sfgf gene deleted and replaced with mgf. Macroscopic, histological and cytological examinations led to the conclusion that growth factors are indeed important for the development and maintenance of fibromas, provided that they are expressed in the proper viral context. However, they are not exchangeable and MGF cannot substitute for SFGF in the genesis of fibromas. It is likely that factors other than viral epidermal growth factor homologues influence the development of tumours.

The Poxvirus p28 Virulence Factor Is an E3 Ubiquitin Ligase

A majority of the orthopoxviruses, including the variola virus that causes the dreaded smallpox disease, encode a highly conserved 28-kDa protein with a classic RING finger sequence motif (C(3)HC(4)) at their carboxyl-terminal domains. The RING domain of p28 has been shown to be a critical determinant of viral virulence for the ectromelia virus (mousepox virus) in a murine infection model (Senkevich, T. G., Koonin, E. V., and Buller, R. M. (1994) Virology 198, 118-128). Here, we demonstrate that the p28 proteins encoded by the ectromelia virus and the variola virus possess E3 ubiquitin ligase activity in biochemical assays as well as in cultured mammalian cells. Point mutations disrupting the RING finger domain of p28 completely abolish its E3 ligase activity. In addition, p28 functions cooperatively with Ubc4 and UbcH5c, the E2 conjugating enzymes involved in 26 S proteasome degradation of protein targets. Moreover, p28 catalyzes the formation of Lys-63-linked polyubiquitin chains in the presence of Ubc13/Uev1A, a heterodimeric E2 conjugating enzyme, indicating that p28 may regulate the biological activity of its cognate viral and/or host cell target(s) by Lys-63-linked ubiquitin multimers. We thus conclude that the poxvirus p28 virulence factor is a new member of the RING finger E3 ubiquitin ligase family and has a unique polyubiquitylation activity. We propose that the E3 ligase activity of the p28 virulence factor may be targeted for therapeutic intervention against infections by the variola virus and other poxviruses.