Camelpox Research Articles

Multiplex PCR for rapid diagnosis and differentiation of pox and pox-like diseases in dromedary Camels.

BackgroundPox and pox-like diseases of camels are a group of exanthematous skin conditions that have become increasingly important economically. Three distinct viruses may cause them: camelpox virus (CMLV), camel parapox virus (CPPV) and camelus dromedary papilloma virus (CdPV). These diseases are often difficult to differentiate based on clinical presentation in disease outbreaks. Molecular methods such as PCR targeting species-specific genes have been developed and used to identify these diseases, but not simultaneously in a single tube. Recently, multiplex PCR has gained reputation as a convenient diagnostic method with cost-and timesaving benefits.Methods and resultsIn the present communication, we describe the development, optimization and validation of a multiplex PCR assay able to detect simultaneously the genome of the three viruses in one single test allowing for rapid and efficient molecular diagnosis. The assay was developed based on the evaluation and combination of published and new primer sets and was validated with viral genomic DNA extracted from known virus strains (n = 14) and DNA extracted from homogenized clinical skin specimens (n = 86). The assay detects correctly the target pathogens by amplification of targeted genes, even in case of co-infection. The method showed high sensitivity, and the specificity was confirmed by PCR-product sequencing.ConclusionThis assay provide rapid, sensitive and specific method for identifying three important viruses in specimens collected from dromedary camels with varying clinical presentations.

Golgi Anti-apoptotic Proteins Are Highly Conserved Ion Channels That Affect Apoptosis and Cell Migration

Golgi anti-apoptotic proteins (GAAPs) are multitransmembrane proteins that are expressed in the Golgi apparatus and are able to homo-oligomerize. They are highly conserved throughout eukaryotes and are present in some prokaryotes and orthopoxviruses. Within eukaryotes, GAAPs regulate the Ca(2+) content of intracellular stores, inhibit apoptosis, and promote cell adhesion and migration. Data presented here demonstrate that purified viral GAAPs (vGAAPs) and human Bax inhibitor 1 form ion channels and that vGAAP from camelpox virus is selective for cations. Mutagenesis of vGAAP, including some residues conserved in the recently solved structure of a related bacterial protein, BsYetJ, altered the conductance (E207Q and D219N) and ion selectivity (E207Q) of the channel. Mutation of residue Glu-207 or -178 reduced the effects of GAAP on cell migration and adhesion without affecting protection from apoptosis. In contrast, mutation of Asp-219 abrogated the anti-apoptotic activity of GAAP but not its effects on cell migration and adhesion. These results demonstrate that GAAPs are ion channels and define residues that contribute to the ion-conducting pore and affect apoptosis, cell adhesion, and migration independently.

ST-246 is a key antiviral to inhibit the viral F13L phospholipase, one of the essential proteins for orthopoxvirus wrapping.

ObjectivesST-246 is one of the key antivirals being developed to fight orthopoxvirus (OPV) infections. Its exact mode of action is not completely understood, but it has been reported to interfere with the wrapping of infectious virions, for which F13L (peripheral membrane protein) and B5R (type I glycoprotein) are required. Here we monitored the appearance of ST-246 resistance to identify its molecular target.MethodsVaccinia virus (VACV), cowpox virus (CPXV) and camelpox virus (CMLV) with reduced susceptibility to ST-246 were selected in cell culture and further characterized by antiviral assays and immunofluorescence. A panel of recombinant OPVs was engineered and a putative 3D model of F13L coupled with molecular docking was used to visualize drug–target interaction. The F13L gene of 65 CPXVs was sequenced to investigate F13L amino acid heterogeneity.ResultsAmino acid substitutions or insertions were found in the F13L gene of six drug-resistant OPVs and production of four F13L-recombinant viruses confirmed their role(s) in the occurrence of ST-246 resistance. F13L, but not B5R, knockout OPVs showed resistance to ST-246. ST-246 treatment of WT OPVs delocalized F13L- and B5R-encoded proteins and blocked virus wrapping. Putative modelling of F13L and ST-246 revealed a probable pocket into which ST-246 penetrates. None of the identified amino acid changes occurred naturally among newly sequenced or NCBI-derived OPV F13L sequences.ConclusionsBesides demonstrating that F13L is a direct target of ST-246, we also identified novel F13L residues involved in the interaction with ST-246. These findings are important for ST-246 use in the clinic and crucial for future drug-resistance surveillance programmes.

Pathomolecular Studies on Staphylococcus Species and Pox Virus Causing Skin Lesions in Teat and Udder of Dromedary She-Camels

The current study aimed to isolate and characterize staphylococcus species and poxvirus in skin lesions of teats and udders of she-camels using conventional and molecular techniques with exploration of their histopathological alterations. A total of 82 tissue samples were collected from affected she-camels. Isolation of staphylococci was attained and determined their antibiogram. Isolation of poxvirus (on Vero cell and chorioallantoic membrane) was achieved and identified as camel poxvirus (CMLV) by virus neutraliztion test . Bacterial and viral isolates were confirmed by PCR. Sequencing and viral phylogenic analyses were performed to determine the homology of isolated sequences with other sequences available in GenBank and to identify the probabilities of zoonotic transmission. Staphylococci were isolated of which, 11 isolates were S.aureus. The other isolates were S.simulanis, S.hemolyticus and S.carnosus. PCR results confirmed S.aureus isolates by specific amplification of 279bp. Two poxviruses were successfully isolated (with titres of 105.5TCID50 and 104TCID50) and confirmed by PCR by amplification of 881bp. Sequence and phylogenic analyses revealed that the two pox isolates were resemble closely Al-Ahsaa strain (CMLV). Pathologically, the udder of she-camels with poxvirus showed crusts grossly and histopathological results revealed hyperkeratosis, acanthosis with degeneration of prickle cells. Udder affected with staphylococci showed edema with abscess grossly. Histopathology revealed pus, necrotic debris and inflammatory cells in the dermis. In conclusion, Isolation, PCR and sequencing with histopathological examination ensure the presence of (CMLV) and staphylocci in skin of udder and teat of she-camels also, confer high level of diagnostic assays for investigation of the diseases in dromedaries.

Molecular investigation and cultivation of camelpox virus in Iran

Camelpox virus (genus Orthopoxvirus, family Poxviridae) is the etiologic agent of camel pox. The clinical manifestations of this virus range from inapparent infection to mild, moderate and, less commonly, severe systemic infection and death. Following an outbreak of camelpox, samples that were collected from camel flocks suspected to have camelpox in Qom Province in central Iran and Khash city, Sistan and Baluchestan Province and South Khorasan Province in eastern Iran were sent to Razi Vaccine and Serum Research Institute in Mashhad. DNA extraction was performed primarily by the phenol-chloroform method, and PCR was carried out using a Bioneer kit. Using the primer pair 5'-AAT-ACA-AGG-AGG-ATC-T-3' and 5'-CTT-AAC-TTT-TTC-TTT-CTC-3', the gene sequence encoding the A-type inclusion protein (ATIP) was amplified. The size of the PCR product, specific for camelpox virus, was 881 bp. The PCR product was purified, and to confirm its sequence, it was sent to the reference laboratory. The sequence was subjected to a BLAST search and then phylogenetically analyzed using CLC software. The results showed that all samples were nearly 100 % identical to each other and to strains CMS and M-96. These isolates also had 99 % and 95 % similarity to the CP-1 strain and isolate FIN/T2000, respectively. In Vero cell culture, inoculation with this virus caused a cytopathic effect (CPE), which appeared 2-5 days post-inoculation. Characteristic CPE showing foci of rounded cells, ballooning, giant-cell formation and syncytia with degenerative changes appeared.

Isolation of Camelpox Virus in Egypt

Isolation of Camelpox Virus in Egypt

Sequence Analysis of Camelpox virus Isolated in Egypt

Sequence Analysis of Camelpox virus Isolated in Egypt

Prediction of Steps in the Evolution of Variola Virus Host Range

Variola virus, the agent of smallpox, has a severely restricted host range (humans) but a devastatingly high mortality rate. Although smallpox has been eradicated by a World Health Organization vaccination program, knowledge of the evolutionary processes by which human super-pathogens such as variola virus arise is important. By analyzing the evolution of variola and other closely related poxviruses at the level of single nucleotide polymorphisms we detected a hotspot of genome variation within the smallpox ortholog of the vaccinia virus O1L gene, which is known to be necessary for efficient replication of vaccinia virus in human cells. These mutations in the variola virus ortholog and the subsequent loss of the functional gene from camelpox virus and taterapox virus, the two closest relatives of variola virus, strongly suggest that changes within this region of the genome may have played a key role in the switch to humans as a host for the ancestral virus and the subsequent host-range restriction that must have occurred to create the phenotype exhibited by smallpox.

TaqMan based real-time duplex PCR for simultaneous detection and quantitation of capripox and orf virus genomes in clinical samples

A rapid and sensitive TaqMan based real-time duplex PCR (drt-PCR) assay for simultaneous detection, differentiation and quantitation of Capripoxvirus (CaPV) and Orf virus (ORFV) DNA, was optimized targeting the highly conserved DNA polymerase genes of these virus genomes. Two pairs of oligonucleotide primers and two hybridization probes labeled with Cy5/BHQ1 and Hex/BHQ1 for CaPV and ORFV, respectively, were used in the drt-PCR assay. The assay was found to be specific only to targeted viruses and did not react with buffalopox virus (BPXV), camelpox virus (CMLV) (Orthopoxviruses) and cDNA of Peste des petits ruminants virus and bluetongue virus, the other common viruses of sheep and goats. The detection limit of the assay was 20 copies for each of the standard plasmid and 35fg of viral genomic DNA for CaPV and ORFV, respectively, in a single and mixed virus population. Both intra—(0.49–4.6% and 0.7–3.7%) and inter—(0.6–2.35% and 0.27–2.1%) assay variations of drt-PCR for CaPV and ORFV DNA were within the acceptable limits, implying high reproducibility and repeatability of the assay. Further, the diagnostic specificity and the sensitivity of the assay was assessed using known virus isolates of sheeppox virus (SPPV), goatpox virus (GTPV) and ORFV and the clinical specimens from sheep and goats. The developed drt-PCR assay was able to detect, differentiate, quantify simultaneously and also to identity mixed infections of CaPV and ORFV in sheep and goats.

Therapeutic Management of Camel Pox - A Case report

Therapeutic Management of Camel Pox - A Case report

Cytopathogenicity of buffalopox and camelpox virus in buffalo fibroblast cells

The primary cultures of fibroblast cells were established from the ear marginal tissue of a young calf of buffalo (Bubalus bubalis). Subsequent passages of primary fibroblast cells were carried out upon 80–90% confluency. The cells were found to be chromosomally stable and free of Mycoplasma contamination. Housekeeping genes namely- GAPDH and β-Actin were amplified from fibroblast cells. To check the utility of developed fibroblast cells in adaptation of virus, cell monolayers were infected with buffalopox virus (BPXV) and camelpox virus (CMLV) isolates, which showed typical virus-specific CPE. The infection was confirmed by PCR amplification of BPXV and CMLV-specific region of C18L gene. The cell line thus developed could be of immense potential in propagation of viruses adopting skin route of infection and their immuno-modulatory associations.

Genome-Wide Comparison of Cowpox Viruses Reveals a New Clade Related to Variola Virus

Zoonotic infections caused by several orthopoxviruses (OPV) like monkeypox virus or vaccinia virus have a significant impact on human health. In Europe, the number of diagnosed infections with cowpox viruses (CPXV) is increasing in animals as well as in humans. CPXV used to be enzootic in cattle; however, such infections were not being diagnosed over the last decades. Instead, individual cases of cowpox are being found in cats or exotic zoo animals that transmit the infection to humans. Both animals and humans reveal local exanthema on arms and legs or on the face. Although cowpox is generally regarded as a self-limiting disease, immunosuppressed patients can develop a lethal systemic disease resembling smallpox. To date, only limited information on the complex and, compared to other OPV, sparsely conserved CPXV genomes is available. Since CPXV displays the widest host range of all OPV known, it seems important to comprehend the genetic repertoire of CPXV which in turn may help elucidate specific mechanisms of CPXV pathogenesis and origin. Therefore, 22 genomes of independent CPXV strains from clinical cases, involving ten humans, four rats, two cats, two jaguarundis, one beaver, one elephant, one marah and one mongoose, were sequenced by using massive parallel pyrosequencing. The extensive phylogenetic analysis showed that the CPXV strains sequenced clearly cluster into several distinct clades, some of which are closely related to Vaccinia viruses while others represent different clades in a CPXV cluster. Particularly one CPXV clade is more closely related to Camelpox virus, Taterapox virus and Variola virus than to any other known OPV. These results support and extend recent data from other groups who postulate that CPXV does not form a monophyletic clade and should be divided into multiple lineages.

Emerging viral diseases of livestock in the developing world

Emerging and reemerging viral diseases of livestock and human beings are in sharp rise in recent years. Importantly, many of these viruses, including influenza, Hendra, Nipah and corona are of zoonotic importance. Several viral diseases of livestock such as bluetongue, peste des petits ruminants, camel pox, equine infectious anaemia, chicken anaemia and sheep-associated malignant catarrhal fever are crossing their traditional boundaries. Emergence of new serotypes and variant forms of viruses as in the case of blue tongue virus, avian infectious bronchitis virus, Newcastle disease virus adds additional level of complexity. The increased incidence of emerging and reemerging viral diseases could be attributed to several factors including deforestation and surge in direct contact of livestock and humans with wild animals and birds. This special issue of “Indian Journal of Virology” is focused on diverse aspects of above diseases: isolation and characterization of viruses, epidemiology, pathogenesis, diagnosis, prevention measures and vaccine development.

KAY-2-41, a Novel Nucleoside Analogue Inhibitor of OrthopoxvirusesIn VitroandIn Vivo

The availability of adequate treatments for poxvirus infections would be valuable not only for human use but also for veterinary use. In the search for novel antiviral agents, a 1'-methyl-substituted 4'-thiothymidine nucleoside, designated KAY-2-41, emerged as an efficient inhibitor of poxviruses. In vitro, KAY-2-41 was active in the micromolar range against orthopoxviruses (OPVs) and against the parapoxvirus orf. The compound preserved its antiviral potency against OPVs resistant to the reference molecule cidofovir. KAY-2-41 had no noticeable toxicity on confluent monolayers, but a cytostatic effect was seen on growing cells. Genotyping of vaccinia virus (VACV), cowpox virus, and camelpox virus selected for resistance to KAY-2-41 revealed a nucleotide deletion(s) close to the ATP binding site or a nucleotide substitution close to the substrate binding site in the viral thymidine kinase (TK; J2R) gene. These mutations resulted in low levels of resistance to KAY-2-41 ranging from 2.7- to 6.0-fold and cross-resistance to 5-bromo-2'-deoxyuridine (5-BrdU) but not to cidofovir. The antiviral effect of KAY-2-41 relied, at least in part, on activation (phosphorylation) by the viral TK, as shown through enzymatic assays. The compound protected animals from disease and mortality after a lethal challenge with VACV, reduced viral loads in the serum, and abolished virus replication in tissues. In conclusion, KAY-2-41 is a promising nucleoside analogue for the treatment of poxvirus-induced diseases. Our findings warrant the evaluation of additional 1'-carbon-substituted 4'-thiothymidine derivatives as broad-spectrum antiviral agents, since this molecule also showed antiviral potency against herpes simplex virus 1 in earlier studies.

Analysis of TK and C18L genes of wild-type and cell culture passaged camelpox virus

Camelpox is an infectious skin disease of camels caused by camelpox virus (CMLV). It is confined to camel rearing belts of developing countries (Wernery U, et al., 2002). CMLV is classified in the Orthopoxvirus (OPV) genus of the subfamily Chordopoxvirinae in the Poxviridae family.

Camelpox, an emerging orthopox viral disease

Camelpox is considered as emerging public health problem during this decade due to increased reported cases and outbreaks in camels. Camelpox is a contagious, often sporadic, and notifiable skin disease of camelids and is socio-economically significant as it incurs considerable loss in terms of morbidity, mortality, loss of weight and reduction in milk yield and confined to camel-rearing countries. The causative agent, camelpox virus (CMLV) is genetically closely related to variola virus and has gained much attention from researchers due to its recent emergence in human. The virus carrying genes responsible for host immune evasion mechanisms owing to the threat posed by potential bio-warfare agents. Although the disease can be diagnosed based on clinical features, the similar confounding skin lesions necessitate identification, detection and differentiation of the CMLV by molecular techniques. Vaccines are available in some countries and the available live attenuated vaccine provides long-lasting immunity. Further, novel highly sensitive and specific techniques would be useful in the identification of emerging and re-emerging virus, thereby therapeutic, prophylactic, preventive measures would be applied in time to curtail further spread of camelpox like other zoonotic diseases. This review provide overview of the camelpox particularly on its epidemiology, pathogenesis and biology of the disease, diagnostic approaches and control measures.

In vitro and in silico biological studies of novel thiazolo[3,2-a]pyrimidine-6-carboxylate derivatives

In the present study, we report the synthesis, characterization of new series of thiazolo[3,2-a]pyrimidine-6-carboxylate derivatives 3a–f and 4a–f. The newly synthesized compounds were screened for in vitro antimicrobial and antiviral activities. The probable mode of action of these active compounds was determined through in silico docking study by docking the receptor methionyl-tRNA synthetase and human inosine-5′-monophosphate dehydrogenase (IMPDH) for antibacterial and antiviral activities, respectively. Among the compounds, 4c exhibited excellent in vitro antimicrobial activity against all tested strains with binding and docking energies −35.6 and −12.4 kcal/mol, respectively. The antiviral studies were carried out for the selected compounds in which 4a exhibited 73.69 and 54.42 % of inhibition of buffalopox and camelpox viruses, respectively. Furthermore, compound 4a showed minimum docking and binding energy along with the maximum hydrogen/hydrophobic interaction with IMPDH. The study contributes towards identification and screening of potential antimicrobial and antiviral agent’s against the pathogens.

Human and Viral Golgi Anti-apoptotic Proteins (GAAPs) Oligomerize via Different Mechanisms and Monomeric GAAP Inhibits Apoptosis and Modulates Calcium

Golgi anti-apoptotic proteins (GAAPs) are hydrophobic proteins resident in membranes of the Golgi complex. They protect cells from a range of apoptotic stimuli, reduce the Ca(2+) content of intracellular stores, and regulate Ca(2+) fluxes. GAAP was discovered in camelpox virus, but it is highly conserved throughout evolution and encoded by all eukaryote genomes examined. GAAPs are part of the transmembrane Bax inhibitor-containing motif (TMBIM) family that also includes other anti-apoptotic and Ca(2+)-modulating membrane proteins. Most TMBIM members show multiple bands when analyzed by SDS-PAGE, suggesting that they may be oligomeric. However, the molecular mechanisms of oligomerization, the native state of GAAPs in living cells and the functional significance of oligomerization have not been addressed. TMBIM members are thought to have evolved from an ancestral GAAP. Two different GAAPs, human (h) and viral (v)GAAP were therefore selected as models to examine oligomerization of TMBIM family members. We show that both hGAAP and vGAAP in their native states form oligomers and that oligomerization is pH-dependent. Surprisingly, hGAAP and vGAAP do not share the same oligomerization mechanism. Oligomerization of hGAAP is independent of cysteines, but oligomerization of vGAAP depends on cysteines 9 and 60. A mutant vGAAP that is unable to oligomerize revealed that monomeric vGAAP retains both its anti-apoptotic function and its effect on intracellular Ca(2+) stores. In conclusion, GAAP can oligomerize in a pH-regulated manner, and monomeric GAAP is functional.

The first isolation and molecular characterization of camelpox virus in Ethiopia

A cross-sectional study was conducted from November 2011 to April 2012 in Chifra district of Afar and in Jigjiga Zone of Somali Regional States of Ethiopia with the aims of assessing the epidemiology of camelpox and isolate and molecularly characterize the virus. The study included a questionnaire, active disease search and virus isolation and sequencing. A total of 24 (4.50%) and 12 (3.0%) camels in Afar and Jigjiga respectively were found clinically sick of camelpox during the study period. The questionnaire survey indicated that camelpox is the most common disease in the areas in which 125 (96%) of the respondents reported the frequent occurrence of camelpox in their herds especially during rainy season. The PCR result revealed 12 out of 17 tested samples were positive, of which seven of them collected from Jigjiga zone showed the characteristic PCR positive bands of 881bp size fragments while five of the Afar samples gave two faint bands. Ethiopian isolates, specially isolated from Somali have very high identity with comparable sequences of CMLV M-96 from Kazakhstan and CMLV CMS from Iran. Out of the total of 780bp analogous sequences, Ethiopian isolates differ only in two positions, while CMLV-Teheran differed at four nucleotide positions. The successfull isolation and molecular characterization of camelpox virus in Ethiopia, which could help for early diagnosis and control of the disease in the country.

Phylogenetic analysis of immunomodulatory protein genes of camelpoxvirus obtained from India

The haemagglutinin (HA) encoding gene and genes encoding for immunomodulatory proteins i.e., schlafen-like protein, epidermal growth factor and golgi anti apoptotic protein of camelpoxvirus (CMLV) obtained from Indian dromedarian camels were cloned and characterized. In this study, the size of the HA encoding gene obtained from the Indian CMLV is 941 bp which is only partial. Sequence analysis of schlafen-like protein gene revealed that CMLV obtained from India shared 99.6% identity with CMLV-Iran and CMLV-Kazakhstan strains both at nucleotide and amino acid level. The size of epidermal growth factor (EGF) gene of Indian CMLV obtained in this study was 418 bp, which was due to the addition of one cytosine residue position 132 of EGF gene of Indian CMLV. Sequence analysis revealed that the Golgi anti-apoptotic protein (GAAP) of Indian CMLV shared 99.5% sequence identity both at the nucleotide and amino acid level with CMLV-Kazakhstan. Based on the nucleotide and amino acid sequence identities and phylogenetic analyses of these genes, it is found that CMLV-India is forming a cluster with Kazakhstan and Iranian CMLV isolates.