Identification, cellular tropism and in vitro transforming properties of ovine papillomaviruses

To date, about 150 animal papillomaviruses (PV) have been identified in benign and malignant lesions of the skin and mucosa in 75 vertebrate host species. In sheep, three ovine PV types have been described so far, namely OaPV1, OaPV2, and OaPV3. All of them infect skin epithelium, but while OaPV1, OaPV2 belong to the Delta-genus and cause benign fibropapillomas, OaPV3 is part of the distant Dyokappa-genus and has been detected in squamous cell carcinomas (SCC). This research aims to identify and genetically characterize novel ovine papillomaviruses types, and to investigate their role in skin carcinogenesis. The identification of OaPV4, a novel ovine PV type within Deltapapillomaviruses 3 in a fibropapilloma of a ram is reported. In vitro transforming abilities of OaPV3 and OaPV4 E6 and E7 PV oncogenes are evaluated in a comparative study to investigate a potential role of Delta and Dyokappa viruses in non-melanoma skin cancer development. Both OaPV3 and OaPV4 E6 and E7 transduction alter cell growth profile of primary human and sheep keratinocytes, but while OaPV4-E6E7 determine a strong lifespan increase OaPV3-E6E7 are able to promote immortalization. Furthermore, pRB protein levels are altered upon E6 and E7 expression, and pulldown assays reveal stronger ability of OaPV3-E7 to efficiently associate with pRB leading to its destabilization. Results support the existence of peculiar in vitro transformation properties for ovine papillomaviruses reinforcing the thesis of a direct link between cutaneous papillomaviruses, cellular transformation, and NMSC progression

Regressing Multiple Viral Plaques and Skin Fragility Syndrome in a Cat Coinfected with FcaPV2 and FcaPV3

Feline viral plaques are uncommon skin lesions clinically characterized by multiple, often pigmented, and slightly raised lesions. Numerous reports suggest that papillomaviruses (PVs) are involved in their development. Immunosuppressed and immunocompetent cats are both affected, the biological behavior is variable, and the regression is possible but rarely documented. Here we report a case of a FIV-positive cat with skin fragility syndrome and regressing multiple viral plaques in which the contemporary presence of two PV types (FcaPV2 and FcaPV3) was demonstrated by combining a quantitative molecular approach to histopathology. The cat, under glucocorticoid therapy for stomatitis and pruritus, developed skin fragility and numerous grouped slightly raised nonulcerated pigmented macules and plaques with histological features of epidermal thickness, mild dysplasia, and presence of koilocytes. Absolute quantification of the viral DNA copies (4555 copies/microliter of FcaPV2 and 8655 copies/microliter of FcaPV3) was obtained. Eighteen months after discontinuation of glucocorticoid therapy skin fragility and viral plaques had resolved. The role of the two viruses cannot be established and it remains undetermined how each of the viruses has contributed to the onset of VP; the spontaneous remission of skin lesions might have been induced by FIV status change over time due to glucocorticoid withdraw and by glucocorticoids withdraw itself

A field study evaluating the humoral immune response in Mongolian sheep vaccinated against sheeppox virus

Sheeppox is a transboundary disease of sheep caused by infection with the capripoxvirus sheeppox virus (SPPV). Sheeppox is found in Africa, the Middle East and Asia and is characterised by fever, multifocal cutaneous raised lesions, and death, with substantial negative impact on affected flocks. Vaccination with live attenuated capripoxvirus (CPPV) strains is an effective and widely used means of controlling sheeppox outbreaks, however there are few reports of post-vaccination field surveillance studies of sheeppox. This study used a commercially available ELISA and a fluorescence-based neutralisation assay (FVNT) to examine quantitative and temporal features of the humoral response of sheep vaccinated with a live attenuated CPPV strain in Mongolia. 400 samples were tested using the ELISA, and a subset of 45 also tested with the FVNT. There was substantial agreement between the FVNT and ELISA tests. Antibodies to CPPV were detected between 40 and 262 days post vaccination. There was no significant difference between serological status (positive / negative) and sex or age, however an inverse correlation was found between the length of time since vaccination and serological status. Animals between 90 and 180 days post-vaccination were more likely to be positive than animals greater than 180 days post vaccination. This data provides temporal parameters to consider when planning sheeppox post-vaccination monitoring programmes. In summary, our results show a commercial CPPV ELISA kit is a robust and reliable assay for use in resource-restricted low and low-middle income countries for post CPPV vaccination surveillance on a regional or national level.The attached .xls file contains all raw data used in the associated publication, " A comparative serological field study evaluating the humoral immune response in Mongolian sheep vaccinated against sheeppox virus." The dataset identifies all individual sheep by a unique identification number (ID_num). Each unique ID_num is associated with relevant metadata: Province name, Sum name, herder name coded (Herded_Id), animal species, age of the animal, in years, when sampled (Age), sex of the animal "F" or "M" (Sex), date when the animal was sampled (Date_sample_collected), date when the animal was vaccinated for sheeppox according to the vaccination records (Date_vaccinated_raw data), and for samples in which the exact date of vaccination was not available and a range of potential time was given, the midpoint date within this range (Date_vaccinated_midpoint), animals for which vaccination date was not available receive an "NA" value; time from vaccination to sampled in days (Time_from_vaccinated_midpoint); %S/P values for the ELISA test conducted in the Mongolia lab (Ag_ELISA_SCVL_OD_SP) and %S/P values for the ELISA test conducted in the UK lab (Ag_ELISA_TPI_OD_SP), results from the ELISA test classified as binary variable "Positive" or "Negative" (Ag_ELISA_SCVL_bin and Ag_ELISA_TPI_bin); titres from the fluorescence-based neutralisation assay (FVNT Titre) and results from the FVNT test classified as binary variable ("Positive" or "Negative"), all animals that were not tested by FVNT receive an "NA" value in these columns. Funding provided by: Biotechnology and Biological Sciences Research CouncilCrossref Funder Registry ID: http://dx.doi.org/10.13039/501100000268Award Number: BBS/E/I/00007031Funding provided by: Biotechnology and Biological Sciences Research CouncilCrossref Funder Registry ID: http://dx.doi.org/10.13039/501100000268Award Number: BB/E/I/00007036Funding provided by: Biotechnology and Biological Sciences Research CouncilCrossref Funder Registry ID: http://dx.doi.org/10.13039/501100000268Award Number: BB/E/I/00007037Funding provided by: Biotechnology and Biological Sciences Research CouncilCrossref Funder Registry ID: http://dx.doi.org/10.13039/501100000268Award Number: BBS/E/I/00007039Funding provided by: Biotechnology and Biological Sciences Research CouncilCrossref Funder Registry ID: http://dx.doi.org/10.13039/501100000268Award Number: BB/J004324/1Funding provided by: Biotechnology and Biological Sciences Research CouncilCrossref Funder Registry ID: http://dx.doi.org/10.13039/501100000268Award Number: BBS/E/D/20002173Funding provided by: Horizon 2020Crossref Funder Registry ID: http://dx.doi.org/10.13039/501100007601Award Number: 773701Funding provided by: Food and Agriculture Organization of the United Nations*Crossref Funder Registry ID: Award Number: TCP/MON/3603Funding provided by: IdVet*Crossref Funder Registry ID: Award Number: Funding provided by: Food and Agriculture Organization of the United NationsCrossref Funder Registry ID: Award Number: TCP/MON/3603Funding provided by: IdVetCrossref Funder Registry ID:Blood samples from sheep and associated data were collected as part of the post-vaccination surveillance programme for sheeppox implemented by the Mongolian General Authority for Veterinary Services (GAVS) in 2016

<i>Mycoplasma agalactiae</i> MAG_5040 is a Mg²⁺-dependent, sugar-nonspecific SNase recognised by the host humoral response during natural infection

In this study the enzymatic activity of Mycoplasma agalactiae MAG_5040, a magnesium-dependent nuclease homologue to the staphylococcal SNase was characterized and its antigenicity during natural infections was established. A UGA corrected version of MAG_5040, lacking the region encoding the signal peptide, was expressed in Escherichia coli as a GST fusion protein. Recombinant GST-MAG_5040 exhibits nuclease activity similar to typical sugar-nonspecific endo- and exonucleases, with DNA as the preferred substrate and optimal activity in the presence of 20 mM MgCl2 at temperatures ranging from 37 to 45uC. According to in silico analyses, the position of the gene encoding MAG_5040 is consistently located upstream an ABC transporter, in most sequenced mycoplasmas belonging to the Mycoplasma hominis group. In M. agalactiae, MAG_5040 is transcribed in a polycistronic RNA together with the ABC transporter components and with MAG_5030, which is predicted to be a sugar solute binding protein by 3D modeling and homology search. In a natural model of sheep and goats infection, anti-MAG_5040 antibodies were detected up to 9 months post infection. Taking into account its enzymatic activity, MAG_5040 could play a key role in Mycoplasma agalactiae survival into the host, contributing to host pathogenicity. The identification of MAG_5040 opens new perspectives for the development of suitable tools for the control of contagious agalactia in small ruminants

Madin-Darby bovine kidney (MDBK) cells are a suitable cell line for the propagation and study of the bovine poxvirus lumpy skin disease virus

Lumpy skin disease virus (LSDV) is a poxvirus that causes systemic disease in cattle, resulting in substantial economic loss to affected communities. LSDV is a rapidly emerging pathogen of growing global concern that recently spread from Africa and the Middle East into Europe and Asia, impacting the cattle population in these regions. An increase in research efforts into LSDV is required to address key knowledge gaps, however this is hampered by lack of suitable cell lines on which to propagate and study the virus. In this work we describe the replication and spread of LSDV on Madin-Darby bovine kidney (MDBK) cells, and the formation of foci-type poxvirus plaques by LSDV on MDBK cells. Methods utilising MDBK cells to quantify neutralising antibodies to LSDV, and to purify LSDV genomic DNA suitable for short read sequencing are described. These research methods broaden the tools available for LSDV researchers and will facilitate the gathering of evidence to underpin the development of LSD control and prevention programmes

Sequencing and Analysis of Lumpy Skin Disease Virus Whole Genomes Reveals a New Viral Subgroup in West and Central Africa

Lumpy skin disease virus (LSDV) is a member of the capripoxvirus (CPPV) genus of the Poxviridae family. LSDV is a rapidly emerging, high-consequence pathogen of cattle, recently spreading from Africa and the Middle East into Europe and Asia. We have sequenced the whole genome of historical LSDV isolates from the Pirbright Institute virus archive, and field isolates from recent disease outbreaks in Sri Lanka, Mongolia, Nigeria and Ethiopia. These genome sequences were compared to published genomes and classified into different subgroups. Two subgroups contained vaccine or vaccine-like samples ("Neethling-like" clade 1.1 and "Kenya-like" subgroup, clade 1.2.2). One subgroup was associated with outbreaks of LSD in the Middle East/Europe (clade 1.2.1) and a previously unreported subgroup originated from cases of LSD in west and central Africa (clade 1.2.3). Isolates were also identified that contained a mix of genes from both wildtype and vaccine samples (vaccine-like recombinants, grouped in clade 2). Whole genome sequencing and analysis of LSDV strains isolated from different regions of Africa, Europe and Asia have provided new knowledge of the drivers of LSDV emergence, and will inform future disease control strategies.</p

Regressing Multiple Viral Plaques and Skin Fragility Syndrome in a Cat Coinfected with FcaPV2 and FcaPV3

Feline viral plaques are uncommon skin lesions clinically characterized by multiple, often pigmented, and slightly raised lesions. Numerous reports suggest that papillomaviruses (PVs) are involved in their development. Immunosuppressed and immunocompetent cats are both affected, the biological behavior is variable, and the regression is possible but rarely documented. Here we report a case of a FIV-positive cat with skin fragility syndrome and regressing multiple viral plaques in which the contemporary presence of two PV types (FcaPV2 and FcaPV3) was demonstrated by combining a quantitative molecular approach to histopathology. The cat, under glucocorticoid therapy for stomatitis and pruritus, developed skin fragility and numerous grouped slightly raised nonulcerated pigmented macules and plaques with histological features of epidermal thickness, mild dysplasia, and presence of koilocytes. Absolute quantification of the viral DNA copies (4555 copies/microliter of FcaPV2 and 8655 copies/microliter of FcaPV3) was obtained. Eighteen months after discontinuation of glucocorticoid therapy skin fragility and viral plaques had resolved. The role of the two viruses cannot be established and it remains undetermined how each of the viruses has contributed to the onset of VP; the spontaneous remission of skin lesions might have been induced by FIV status change over time due to glucocorticoid withdraw and by glucocorticoids withdraw itself

Molecular detection and identification of Rickettsiales pathogens in dog ticks from Costa Rica

Although vector-borne diseases are globally widespread with considerable impact on animal production and on public health, few reports document their presence in Central America. This study focuses on the detection and molecular identification of species belonging to selected bacterial genera (Ehrlichia, Anaplasma and Rickettsia) in ticks sampled from dogs in Costa Rica by targeting several genes: 16S rRNA/dsb genes for Ehrlichia; 16S rRNA/groEL genes for Anaplasma, and ompA/gltA/groEL genes for Rickettsia. PCR and sequence analyses provides evidences of Ehrlichia canis, Anaplasma platys, and Anaplasma phagocytophilum infection in Rhipicephalus sanguineus s.l ticks, and allow establishing the presence of Rickettsia monacensis in Ixodes boliviensis. Furthermore, the presence of recently discovered Mediterranean A. platys-like strains is reported for the first time in Central America. Results provide new background on geographical distribution of selected tick-transmitted bacterial pathogens in Costa Rica and on their molecular epidemiology, and are pivotal to the development of effective and reliable diagnostic tools in Central America.Aunque las enfermedades transmitidas por vectores están generalizadas en todo el mundo con un impacto considerable en la producción animal y en la salud pública, pocos informes documentan su presencia en América Central. Este estudio se enfoca en la detección e identificación molecular de especies pertenecientes a géneros bacterianos seleccionados (Ehrlichia, Anaplasma y Rickettsia) en garrapatas muestreadas de perros en Costa Rica mediante el objetivo de varios genes: 16S rRNA / dsb genes para Ehrlichia; 16S rRNA / groEL genes para Anaplasma, y ​​ompA / gltA / groEL genes para Rickettsia. La PCR y los análisis de secuencia proporcionan evidencias de infección por Ehrlichia canis, Anaplasma platys y Anaplasma phagocytophilum en garrapatas Rhipicephalus sanguineus s.l, y permiten establecer la presencia de Rickettsia monacensis en Ixodes boliviensis. Además, la presencia de cepas mediterráneas recientemente descubiertas tipo A. platys se informa por primera vez en América Central. Los resultados proporcionan nuevos antecedentes sobre la distribución geográfica de patógenos bacterianos transmitidos por garrapatas seleccionados en Costa Rica y sobre su epidemiología molecular, y son fundamentales para el desarrollo de herramientas de diagnóstico efectivas y confiables en América Central.Universidad Nacional, Costa RicaEscuela de Medicina Veterinari

Leopardus wiedii Papillomavirus type 1, a novel papillomavirus species in the tree ocelot, suggests Felidae Lambdapapillomavirus polyphyletic origin and host-independent evolution

The limited knowledge on Papillomavirus diversity (particularly in wild animal species) influences the accuracy of PVs phylogeny and their evolutionary history, and hinders the comprehension of PVs pathogenicity, especially the mechanism of virus - related cancer progression. This study reports the identification of Leopardus wiedii Papillomavirus type 1 (LwiePV1), the first PV type within Lambdapapillomavirus in a Leopardus host. LwiePV1 full genome sequencing allowed the investigation of its taxonomic position and phylogeny. Based on results, LwiePV1 should be assigned to a novel PV species providing evidence for a polyphyletic origin of feline lambda PVs, and representing an exception to codivergence between feline lambda PVs and their hosts. Results improve our knowledge on PV diversity and pave the way to future studies investigating biological and evolutionary features of animal PVs.El conocimiento limitado sobre la diversidad del virus del papiloma (particularmente en especies de animales salvajes) influye en la precisión de la filogenia de los PVs y su historia evolutiva, y dificulta la comprensión de la patogenicidad de los PVs, especialmente el mecanismo de progresión del cáncer relacionado con el virus. Este estudio informa la identificación del virus del papiloma de Leopardus wiedii tipo 1 (LwiePV1), el primer tipo de PV dentro del virus del papiloma de Lambda en un huésped Leopardus. La secuenciación completa del genoma de LwiePV1 permitió la investigación de su posición taxonómica y filogenia. Según los resultados, LwiePV1 debe asignarse a una nueva especie de PV que proporcione evidencia de un origen polifilético de PVs lambda felina y que represente una excepción a la codivergencia entre PVs lambda felina y sus huéspedes. Los resultados mejoran nuestro conocimiento sobre la diversidad PVs y allanan el camino para futuros estudios que investigan las características biológicas y evolutivas de los animales PVs .Universidad Nacional, Costa Ric

Mycoplasma agalactiae MAG_5040 is a Mg2+-dependent, sugar-nonspecific SNase recognised by the host humoral response during natural infection.

In this study the enzymatic activity of Mycoplasma agalactiae MAG_5040, a magnesium-dependent nuclease homologue to the staphylococcal SNase was characterized and its antigenicity during natural infections was established. A UGA corrected version of MAG_5040, lacking the region encoding the signal peptide, was expressed in Escherichia coli as a GST fusion protein. Recombinant GST-MAG_5040 exhibits nuclease activity similar to typical sugar-nonspecific endo- and exonucleases, with DNA as the preferred substrate and optimal activity in the presence of 20 mM MgCl2 at temperatures ranging from 37 to 45°C. According to in silico analyses, the position of the gene encoding MAG_5040 is consistently located upstream an ABC transporter, in most sequenced mycoplasmas belonging to the Mycoplasma hominis group. In M. agalactiae, MAG_5040 is transcribed in a polycistronic RNA together with the ABC transporter components and with MAG_5030, which is predicted to be a sugar solute binding protein by 3D modeling and homology search. In a natural model of sheep and goats infection, anti-MAG_5040 antibodies were detected up to 9 months post infection. Taking into account its enzymatic activity, MAG_5040 could play a key role in Mycoplasma agalactiae survival into the host, contributing to host pathogenicity. The identification of MAG_5040 opens new perspectives for the development of suitable tools for the control of contagious agalactia in small ruminants