Camelpox Research Articles

A review of chemical, biological and fertility control options for the camel in Australia

Since their introduction to Australia in 1840 the one-humped camel, Camelus dromedarius, has gone from the colonist’s companion to a conservationist’s conundrum in the fragile arid ecosystems of Australia. Current management techniques are failing to curb present population growth and alternatives must be sought. This review assess the applicability of currently registered and developmental vertebrate pesticides and fertility control agents for camel control, as well as examining the potential usefulness of known C. dromedarius diseases for biological control. Not surprisingly, little is known about the lethality of most vertebrate pesticides used in Australia to camels. More has been published on adverse reactions to pharmaceuticals used in agriculture and the racing industry. An examination of the literature on C. dromedarius diseases, such as camel pox virus, contagious ecthyma and papillomatosis, indicates that the infections generally result in high morbidity but not necessarily mortality and this alone may not justify their consideration for use in Australia. The possibility exists that other undiscovered or unstudied biological control agents from other camilid species may offer greater potential for population control. As a long-lived species the camel is also not ideally suited to fertility control. Notwithstanding, anti-fertility agents may have their place in preventing the re-establishment of camel populations once they have been reduced through mechanical, biological or chemical means. Delivery of any generic chemical or fertility control agent will, however, require a species-tailored pathway and an appropriate large-scale deployment method. Accordingly, we put forward avenues of investigation to yield improved tools for camel control.

Detection of camel pox and vaccinia viruses by polymerase chain reaction.

PCR following two methods of DNA extraction was used to confirm the growth of camel pox virus (CPV) and vaccinia virus in cell culture and chorioallantoic membrane. Results were compared with the commonly used neutralization test. The first method of DNA extraction was accomplished by using viral DNA in tissue culture supernatant and Chorioallantoic membrane, which was released by initial heating for 15 min at 99 degrees C followed by ordinary PCR. In the second method DNA was extracted by using DNA Isolation Kit from tissue culture supernatant and used as a template. Rapid identification and differentiation of CPV and Vaccinia virus were achieved by PCR and this assay proved to be fast and feasible, and can be an alternative to orthodox serological methods.

A Polymerase Chain Reaction Strategy for the Diagnosis of Camelpox

Camelpox is a contagious viral skin disease that is mostly seen in young camels. The disease is caused by the Camelpox virus (CMLV). In the present study, a polymerase chain reaction (PCR) assay based on the C18L gene (encoding ankyrin repeat protein) and a duplex PCR based on the C18L and DNA polymerase (DNA pol) genes were developed. The former assay yields a specific amplicon of 243 bp of the C18L gene, whereas the duplex PCR yields 243- and 96-bp products of the C18L and DNA pol genes, respectively, in CMLV, and only a 96-bp product of the DNA pol gene in other orthopoxviruses. The limit of detection was as low as 0.4 ng of viral DNA. Both PCR assays were employed successfully for the direct detection and differentiation of CMLV from other orthopoxviruses, capripoxviruses, and parapoxviruses in both cell culture samples and clinical material. Furthermore, a highly sensitive SYBR Green dye-based, real-time PCR was optimized for quantitation of CMLV DNA. In the standard curve of the quantitative assay, the melting temperature of the specific amplicon at 77.6 degrees C with peak measured fluorescence in dissociation plot was observed with an efficiency of 102%. To the authors' knowledge, this is the first report to describe a C18L gene-based PCR for specific diagnosis of camelpox infection.

Detection of human orthopoxvirus infections and differentiation of smallpox virus with real‐time PCR

We developed a real-time PCR protocol to detect orthopoxviruses (OPVs) from different clinical specimens and to separate variola virus from other OPVs. In our protocol, we used automated nucleic acid extraction system together with real-time PCR to create a simple, safe and fast procedure to obtain an initial result. The sensitivity was better by using designed hybridization probes as compared to SYBR green I for detection. The detection limit ranged from 13 to 1,300 copies per 20 microl reaction volume depending on the sample type. The PCR detected all OPVs pathogenic to human (variola, cowpox, monkeypox, vaccinia) as well as camelpox and ectromelia viruses. Amplification of variola virus sequences could be distinguished from other OPVs by melting curve analysis. We also demonstrated the applicability of the assay in human cases of cowpox and vaccinia virus infections.

Specific Targeting of the F13L Protein by St-246 Affects Orthopoxvirus Production Differently

ST-246 is a potent anti-orthopoxviral molecule targeting the F13L protein of vaccinia virus, which is involved in the wrapping of viruses. The discrepancy in sensitivities of several orthopoxviruses to ST-246 has raised questions about potential differences in their replicative cycles and/or the presence of another drug target. Density gradients were used to evaluate the differences between the viral cycles of vaccinia, cowpox and camelpox viruses. Also, to investigate if ST-246 inhibits a single target, we compared its activity to that of small interfering RNAs designed to silence the F13L gene (siF13Ls). We showed that the spread of vaccinia virus involved both intracellular and extracellular enveloped viruses, whereas both cowpox and camelpox viruses seemed to propagate via non-enveloped intracellular forms and cell-associated viral particles. Although ST-246 exerted a clear antiviral activity by interfering with the egress of the virus from infected cells, we observed that cowpox and camelpox viruses, in contrast to vaccinia virus, could be directed towards a lytic cycle under ST-246 treatment. We specifically knocked down the F13L transcripts of vaccinia and camelpox viruses by > 85%, reduced virus progeny by 90% and showed that siF13Ls affect camelpox and vaccinia virus propagation differently. Flow cytometry data validated that ST-246 interfered with the activity of the F13L protein, whereas siF13Ls silenced the F13L gene. Our observations support that vaccinia, cowpox and camelpox viruses exhibit different levels of sensitivity to ST-246 because of dissimilarities between their ways of propagation, and provide a better understanding of the mode of action of ST-246.

Dual-probe real-time PCR assay for detection of variola or other orthopoxviruses with dried reagents

A real-time, multiplexed polymerase chain reaction (PCR) assay based on dried PCR reagents was developed. Only variola virus could be specifically detected by a FAM (6-carboxyfluorescein)-labeled probe while camelpox, cowpox, monkeypox and vaccinia viruses could be detected by a TET (6-carboxytetramethylrhodamine)-labeled probe in a single PCR reaction. Approximately 25 copies of cloned variola virus DNA and 50 copies of genomic orthopoxviruses DNA could be detected with high reproducibility. The assay exhibited a dynamic range of seven orders of magnitude with a correlation coefficient value greater than 0.97. The sensitivity and specificity of the assay, as determined from 100 samples that contained nucleic acids from a multitude of bacterial and viral species were 96% and 98%, respectively. The limit of detection, sensitivity and specificity of the assay were comparable to standard real-time PCR assays with wet reagents. Employing a multiplexed format in this assay allows simultaneous discrimination of the variola virus from other closely related orthopoxviruses. Furthermore, the implementation of dried reagents in real-time PCR assays is an important step towards simplifying such assays and allowing their use in areas where cold storage is not easily accessible.

Viral targeting of DEAD box protein 3 reveals its role in TBK1/IKKɛ-mediated IRF activation

Viruses are detected by different classes of pattern recognition receptors (PRRs), such as Toll-like receptors and RIG-like helicases. Engagement of PRRs leads to activation of interferon (IFN)-regulatory factor 3 (IRF3) and IRF7 through IKKepsilon and TBK1 and consequently IFN-beta induction. Vaccinia virus (VACV) encodes proteins that manipulate host signalling, sometimes by targeting uncharacterised proteins. Here, we describe a novel VACV protein, K7, which can inhibit PRR-induced IFN-beta induction by preventing TBK1/IKKepsilon-mediated IRF activation. We identified DEAD box protein 3 (DDX3) as a host target of K7. Expression of DDX3 enhanced Ifnb promoter induction by TBK1/IKKepsilon, whereas knockdown of DDX3 inhibited this, and virus- or dsRNA-induced IRF3 activation. Further, dominant-negative DDX3 inhibited virus-, dsRNA- and cytosolic DNA-stimulated Ccl5 promoter induction, which is also TBK1/IKKepsilon dependent. Both K7 binding and enhancement of Ifnb induction mapped to the N-terminus of DDX3. Furthermore, virus infection induced an association between DDX3 and IKKepsilon. Therefore, this study shows for the first time the involvement of a DEAD box helicase in TBK1/IKKepsilon-mediated IRF activation and Ifnb promoter induction.

Growth characteristic of Camel pox and Vaccinia viruses in embryonated eggs and cell culture

Camel pox viruses isolated in Sudan and VD45 (African camel pox strain) and Vaccinia virus (Elstree strain) were used for inoculation of chorioallantoic membrane (CAM) of embryonated eggs (EE) and cell culture (CC). In EE Lesions were seen as pocks ranging in size from 1 to 1.5 mm in diameter, and they increase in size with serial passage and taking opaque- white and opaque- yellow colors. When propagated in Vero cells, these viruses gave clear CPE, characterized by rounding of cells, plaque formation, syncytia and detachment of cells from the glass.

Effects of Acp26 on in vitro and in vivo productivity, pathogenesis and virulence of Autographa californica multiple nucleopolyhedrovirus

Homologs to Autographa californica multiple nucleopolyhedrovirus (AcMNPV) open reading frame (ORF) 136 or Acp26 are present within almost all nucleopolyhedroviruses (NPVs). Two copies of the gene are found in some members of group II NPVs, suggesting that it may play an important role in transmission or replication. Phylogenetic analysis revealed that the predicted protein has some similarity with Camelpox virus v-slfn protein, which reduces the virulence of orthopoxviruses in vivo. To investigate the influence of ACP26 on the infectivity and virulence of AcMNPV a bacmid system was used to delete the Acp26 ORF. The Acp26null bacmid was able to generate a transmissible infection in cell culture and larvae, indicating that Acp26 is not essential for propagation of viral infection in vitro or in vivo. Deletion of Acp26 from the AcMNPV genome had no apparent effect on timing or production of infectious BV in cell culture or in insect larvae. Additional comparisons of AcMNPV and Acp26null bacmid viruses showed that deletion of Acp26 did not significantly influence the infectivity and virulence of AcMNPV occlusion bodies or the number produced. The Acp26 may be an auxiliary gene with subtle effects on virus replication and transmission.

Antiviral Potency of ST-246 on the Production of Enveloped Orthopoxviruses and Characterization of ST-246 Resistant Vaccinia, Cowpox and Camelpox Viruses

Antiviral Potency of ST-246 on the Production of Enveloped Orthopoxviruses and Characterization of ST-246 Resistant Vaccinia, Cowpox and Camelpox Viruses

A simple restriction fragment PCR approach for discrimination of humanpathogenic Old World animal Orthopoxvirus species

There are reliable polymerase chain reaction assays available for exclusion of Variola virus from other poxviruses. However, the discrimination of humanpathogenic animal Orthopoxviridae is more challenging because of the high genomic conservation. Based on the variability of the A36R gene, we describe a simple 20 min PCR assay followed by a 1 h digest with 3 different restriction enzymes. This assay enables rapid discrimination between Cowpox virus and Monkeypox virus and discrimination of the most prevalent members of the Vaccinia virus and Camelpox virus. The test was orthopoxvirus specificand did not react with parapox (Orf) virus. Moreover, the amplified fragments were also well suited for additional genotyping by direct DNA sequencing.

Activity of the Anti-Orthopoxvirus Compound ST-246 against Vaccinia, Cowpox and Camelpox Viruses in Cell Monolayers and Organotypic Raft Cultures

The potential use of variola virus as a biological weapon has renewed efforts in the development of antiviral agents against orthopoxviruses. ST-246 [4-trifluoromethyl-N-(3,3a,4,4a,5,5a,6,6a-octahydro-1,3-di oxo-4,6-ethenocycloprop [f]isoindol-2(1 H)-yl)-benzamide] is an anti-orthopoxvirus compound active against several orthopoxviruses including vaccinia virus (VV), cowpox virus (CPV), camelpox virus (CMLV), ectromelia virus (ECTV) and variola virus in cell culture. The compound has been shown to inhibit the release of extracellular virus by targeting the F13L W protein and to protect mice from W, CPV and ECTV orthopoxvirus-induced disease. The antiviral activity of ST-246 was assessed against extracellular and intracellular W, CPV and CMLV production in human embryonic lung (HEL) fibroblasts and primary human keratinocyte (PHK) cell monolayers, as well as in three-dimensional raft cultures. ST-246 inhibited preferentially the production of extracellular virus compared with intracellular virus production in HEL and PHK cells (for W) and in PHK cells (for CMLV). In organotypic epithelial raft cultures, ST-246 at 20 microg/ml inhibited extracellular W and CMLV production by 6 logs, whereas intracellular virus yield was reduced by 2 logs. In the case of CPV, both extracellular and intracellular virus production were completely inhibited by ST-246 at 20 microg/ml. Histological sections of the infected rafts, treated with increasing amounts of drug, confirmed the antiviral activity of ST-246: the epithelium was protected and there was no evidence of viral infection. Electron microscopic examination confirmed the absence of intracellular enveloped virus forms in W-, CPV- and CMLV-infected cells treated with 10 microg/ml of ST-246. These data indicate that ST-246 is a potent anti-orthopoxvirus compound; the mode of inhibition is dependent on the virus and cell type.

Activities of Several Classes of Acyclic Nucleoside Phosphonates against Camelpox Virus Replication in Different Cell Culture Models

Camelpox virus (CMLV) is the closest known virus to variola virus. Here we report on the anti-CMLV activities of several acyclic nucleoside phosphonates (ANPs) related to cidofovir [(S)-1-(3-hydroxy-2-phosphonomethoxypropyl)cytosine (HPMPC; Vistide)] against two CMLV strains, CML1 and CML14. Cytopathic effect (CPE) reduction assays performed with human embryonic lung fibroblast monolayers revealed the selectivities of the first two classes of ANPs (cHPMPA, HPMPDAP, and HPMPO-DAPy) and of the hexadecyloxyethyl ester of 1-{[(5S)-2-hydroxy-2-oxido-1,4,2-dioxaphosphinan-5-yl]methyl}-5-azacytosine (HDE-cHPMP-5-azaC), belonging to the newly synthesized ANPs, which are HPMP derivatives containing a 5-azacytosine moiety. The inhibitory activities of ANPs against both strains were also confirmed with primary human keratinocyte (PHK) monolayers, despite the higher toxicity of those molecules on growing PHKs. Virus yield assays confirmed the anti-CML1 and anti-CML14 efficacies of the compounds selected for the highest potencies in CPE reduction experiments. Ex vivo studies were performed with a 3-dimensional model of human skin, i.e., organotypic epithelial raft cultures of PHKs. It was ascertained by histological evaluation, as well as by virus yield assays, that CMLV replicated in the human skin equivalent. HPMPC and the newly synthesized ANPs proved to be effective at protecting the epithelial cells against CMLV-induced CPE. Moreover, in contrast to the toxicity on PHK monolayers, signs of toxicity in the differentiated epithelium were seen only at high ANP concentrations. Our results demonstrate that compounds belonging to the newly synthesized ANPs, in addition to cidofovir, represent promising candidates for the treatment of poxvirus infections.

Poxviruses as possible vectors for horizontal transfer of retroposons from reptiles to mammals.

Poxviruses (Poxviridae) are a family of double-stranded DNA viruses with no RNA stage. Members of the genus Orthopoxvirus (OPV) are highly invasive and virulent. It was recently shown that the taterapox virus (TATV) from a West African rodent is the sister of camelpox virus and therefore belongs to the clade closest to the variola virus (VARV), the etiological agent of smallpox. Although these OPVs are among the most dreaded pathogens on Earth, our current knowledge of their genomes, their origins, and their possible hosts is still very limited. Here, we report the horizontal transfer of a retroposon (known only from reptilian genomes) to the TATV genome. After isolating and analyzing different subfamilies of short interspersed elements (SINEs) from lizards and snakes, we identified a highly poisonous snake (Echis ocellatus) from West Africa as the closest species from which the SINE sequence discovered in the TATV genome (TATV-SINE) was transferred to the virus. We discovered direct repeats derived from the virus flanking the TATV-SINE, and the absence of any snake-derived DNA flanking the SINE. These data provide strong evidence that the TATV-SINE was actually transferred within the snake to the viral genome by retrotransposition and not by any horizontal transfer at the DNA level. We propose that the snake is another host for TATV, suggesting that VARV-related epidemiologically relevant viruses may have derived from our cold-blooded ancestors and that poxviruses are possible vectors for horizontal transfer of retroposons from reptiles to mammals.

Camelpox virus encodes a schlafen-like protein that affects orthopoxvirus virulence.

Camelpox virus (CMLV) gene 176R encodes a protein with sequence similarity to murine schlafen (m-slfn) proteins. In vivo, short and long members of the m-slfn family inhibited T-cell development, whereas in vitro, only short m-slfns caused arrest of fibroblast growth. CMLV 176 protein (v-slfn) is most closely related to short m-slfns; however, when expressed stably in mammalian cells, v-slfn did not inhibit cell growth. v-slfn is a predominantly cytoplasmic 57 kDa protein that is expressed throughout infection. Several other orthopoxviruses encode v-slfn proteins, but the v-slfn gene is fragmented in all sequenced variola virus and vaccinia virus (VACV) strains. Consistent with this, all 16 VACV strains tested do not express a v-slfn detected by polyclonal serum raised against the CMLV protein. In the absence of a small animal model to study CMLV pathogenesis, the contribution of CMLV v-slfn to orthopoxvirus virulence was studied via its expression in an attenuated strain of VACV. Recombinant viruses expressing wild-type v-slfn or v-slfn tagged at its C terminus with a haemagglutinin (HA) epitope were less virulent than control viruses. However, a virus expressing v-slfn tagged with the HA epitope at its N terminus had similar virulence to controls, implying that the N terminus has an important function. A greater recruitment of lymphocytes into infected lung tissue was observed in the presence of wild-type v-slfn but, interestingly, these cells were less activated. Thus, v-slfn is an orthopoxvirus virulence factor that affects the host immune response to infection.

Real-Time PCR to Identify Variola Virus or Other Human Pathogenic Orthopox Viruses

Variola virus (family Poxviridae, genus Orthopoxvirus) and the closely related cowpox, vaccinia, and monkeypox viruses can infect humans. Efforts are mounting to replenish the smallpox vaccine stocks, optimize diagnostic methods for poxviruses, and develop new antivirals against smallpox, because it is feared that variola virus might be used as a weapon of bioterrorism. We developed an assay for the detection of variola virus DNA. The assay is based on TaqMan chemistry targeting the 14-kD protein gene. For the 1st stage of the assay we used genus consensus primers and a mixture of 2 probes (14-kD POX and 14-kD VAR) spanning the 14-kD protein-encoding gene for detection of all human pathogenic orthopoxviruses. We then tested positive samples with the specific orthopoxvirus-specific probe 14-kD POX to identify monkeypox, cowpox, and vaccinia viruses and with the 14-kD VAR probe to identify variola viruses. The assay was established on 4 different PCR cycler platforms. It was assessed in a study with 85 different orthopoxvirus species and strains that included variola, camelpox, cowpox, monkeypox, and vaccinia viruses at concentrations ranging from 100 ng/L to 1 microg/L. The assay detected as little as 0.05 fg of DNA, corresponding to 25 copies of DNA, and enabled differentiation of variola virus from the other orthopoxviruses. This real-time PCR assay provides a rapid method for the early detection and differentiation of smallpox and other human pathogenic orthopoxvirus infections.

Antiviral Activities of New Cidofovir Analogs Against Camelpox Virus, Used as a Model of Variola Virus, in Human Skin Equivalent Cultures

Antiviral Activities of New Cidofovir Analogs Against Camelpox Virus, Used as a Model of Variola Virus, in Human Skin Equivalent Cultures

A new inhibitor of apoptosis from vaccinia virus and eukaryotes.

A new apoptosis inhibitor is described from vaccinia virus, camelpox virus, and eukaryotic cells. The inhibitor is a hydrophobic, multiple transmembrane protein that is resident in the Golgi and is named GAAP (Golgi anti-apoptotic protein). Stable expression of both viral GAAP (v-GAAP) and human GAAP (h-GAAP), which is expressed in all human tissues tested, inhibited apoptosis induced by intrinsic and extrinsic apoptotic stimuli. Conversely, knockout of h-GAAP by siRNA induced cell death by apoptosis. v-GAAP and h-GAAP display overlapping functions as shown by the ability of v-GAAP to complement for the loss of h-GAAP. Lastly, deletion of the v-GAAP gene from vaccinia virus did not affect virus replication in cell culture, but affected virus virulence in a murine infection model. This study identifies a new regulator of cell death that is highly conserved in evolution from plants to insects, amphibians, mammals, and poxviruses.

APPLICATION OF IMMUNOHISTOCHEMISTRY TO DETECT CAMELPOX VIRUS IN CELL CULTURE AND HISTOLOGICAL SECTIONS

During the spring and summer of 2005, an outbreak of camelpox was spread in the province of Hama and Duma city. Therefore, this study was designed to detect camelpox virus in Vero cell culture and paraffin tissue section by immunohistochemistry. Skin samples (pustules, vesicles and scabs) from infected camels were collected from Duma and Hama. Monoclonal antibody (mouse anti-camelpox IgG) and labeled secondary antibody (rabbit anti-mouse IgG HRP) were used to demonstrate camelpox virus. Camelpox virus was localized in the cytopathic effects in cell culture (syncytia and round cells) and inclusion bodies in the dermal and epidermal cell of the skin. The present study shows that immunohistochemistry is a sensitive diagnostic technique to detect the infection in skin samples with sensitivity of 98% and reflects its effectiveness and accuracy in camelpox infection diagnosis.

The First Outbreak of Camelpox in Syria

In this study we report the first outbreak of camelpox in two provinces in Syria. Clinical symptoms started with fever, salivation and general exanthema. The main features were facial and legs oedema, pustules on the mucosa of the lips and a high rate of abortion. Lesions may also occur on the whole body including scrotum and udder. Specimens were investigated by electron microscopy, virus isolation in cell culture and embryonated eggs and by immunohistochemistry. The causative agent was identified as camelpox virus by polymerase chain reaction and sequencing of the hemagglutinin gene.