Detection, prevalence and molecular epidemiology of virus in cetaceans from Mediterranean sea

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Veterinaria, Departamento de Sanidad Animal, leída el 28-09-2015Las enfermedades infecciosas en cetáceos tienen mucha repercusión en estos animales por las consecuencias que acarrean su presencia. Hasta el momento, sólo se conocen unas pocas, de las cuales las enfermedades víricas ocupan un pequeño porcentaje. Estas enfermedades se pueden dividir en enfermedades emergentes y reemergentes, aunque muchas de ellas han sido recientemente descubiertas. Morbillivirus de los cetáceos, CeMV, es la enfermedad vírica reemergente más letal conocida hasta el momento en cetáceos. Es un virus ARN que afecta fundamentalmente a los sistemas respiratorio, linfático y nervioso de estos animales produciéndoles la muerte. Han sido descritos varios brotes en todo el mundo, dos de ellos localizados en la costa mediterránea española, convirtiéndose en una zona de alto riesgo. Por otro lado, adenovirus, es un virus descrito anteriormente en leones marinos, y que está asociado a hepatitis letales en estos animales. Sin embargo, en cetáceos sólo existen un par de trabajos sobre la asociación de este virus a hisopos y biopsias gastrointestinales pero no se había asociado a ninguna patología hasta ahora. Esta tesis doctoral con el título Detección, prevalencia y epidemiología molecular de virus en cetáceos del Mediterráneo se ha centrado en estos importantes virus de los cetáceos para intentar aportar mejoras en su conocimiento y control. El trabajo desarrollado en la presente tesis, ha generado cuatro artículos científicos publicados en revistas de impacto internacional. Se ha dividido en dos objetivos siendo el primero dividido en tres subobjetivos. Cada uno de ellos corresponde a un artículo científico. El primer objetivo se centra en el avance en el conocimiento de CeMV. El primer subobjetivo trata de la mejora de las técnicas de diagnóstico de esta enfermedad. Se diseñó una PCR a tiempo real para detectar las tres cepas de CeMV, y que a su vez es rápida, sensible, específica y asequible. El segundo subobjetivo se basa en la actuación y diagnóstico ante un brote de CeMV. En este caso se describe el último brote de morbillivirus del delfín, DMV, que aconteció entre marzo y mayo de 2011, en las costas mediterráneas valencianas. Se utilizó la nueva PCR para el diagnóstico de los animales positivos pudiendo avalar la nueva técnica. Y se pudo observar como el 62 por ciento de los animales estudiados fueron positivos a DMV, y en el caso de los delfines listados necropsiados 73 por ciento fueron positivos. El último subobjetivo, se centra en el seguimiento de esta enfermedad en el mar Mediterráneo, entre 2011 y 2014. Se pudo encontrar que de los 177 animales estudiados, sólo tres animales fueron positivos a DMV. En el primero se encontró una infección sistémica causada por una secuencia casi idéntica a la que produjo el brote de 2011, en el segundo y el tercero la secuencia encontrada era idéntica entre sí y diferente a la secuencia del 2011. De hecho, era más parecida a la del brote de 1990 que a la de 2007 y 2011. El segundo objetivo se centra en la detección de nuevos virus en cetáceos, y en este caso de adenovirus. Se pudo asociar por primera vez en cetáceos una gastroenteritis autolimitante con adenovirus. Se hizo el estudio de la enfermedad, viendo que los animales eliminaban el virus hasta 5 días tras la finalización de los síntomas y se consiguió aislar el virus en cultivo celular y confirmarlo mediante inmunofluorescencia y microscopía electrónica. Por último la secuencia obtenida en todos los animales era idéntica entre sí, y parece ser una nueva especie de adenovirus. Finalmente, hay que destacar que esta tesis doctoral aporta una información muy relevante en el campo de los cetáceos, ya que mejora las técnicas de diagnóstico de CeMV, describe un nuevo brote de CeMV y ha ampliado el número de enfermedades víricas que se conocían hasta el momento en estos animalesDepto. de Sanidad AnimalFac. de VeterinariaTRUEunpu

Mecanismo de defensa del erizo europeo (Erinaceus europaeus)

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Tracto digestivo de un insectívoro: erizo europeo (Erinaceus europaeus)

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First molecular determination of herpesvirus from two mysticete species stranded in the Mediterranean Sea

BACKGROUND Herpesvirus can infect a wide range of animal species: mammals, birds, reptiles, fish, amphibians and bivalves. In marine mammals, several alpha- and gammaherpesvirus have been identified in some cetaceans and pinnipeds species. To date, however, this virus has not been detected in any member of the Balaenoptera genus. CASE PRESENTATION Herpesvirus was determined by molecular methods in tissue samples from a male fin whale juvenile (Balaenoptera physalus) and a female common minke whale calf (Balaenoptera acutorostrata) stranded on the Mediterranean coast of the Region of Valencia (Spain). Samples of skin and penile mucosa from the fin whale and samples of skin, muscle and central nervous system tissue from the common minke whale tested positive for herpesvirus based on sequences of the DNA polymerase gene. Sequences from fin whale were identical and belonged to the Alphaherpesvirinae subfamily. Only members of the Gammaherpesvirinae subfamily were amplified from the common minke whale, and sequences from the muscle and central nervous system were identical. Sequences in GenBank most closely related to these novel sequences were viruses isolated from other cetacean species, consistent with previous observations that herpesviruses show similar phylogenetic branching as their hosts. CONCLUSIONS To our knowledge, this is the first molecular determination of herpesvirus in the Balaenoptera genus. It shows that herpesvirus should be included in virological evaluation of these animals

Novel adenovirus detected in captive bottlenose dolphins (Tursiops truncatus) suffering from self-limiting gastroenteritis

BACKGROUND Adenoviruses are common pathogens in vertebrates, including humans. In marine mammals, adenovirus has been associated with fatal hepatitis in sea lions. However, only in rare cases have adenoviruses been detected in cetaceans, where no clear correlation was found between presence of the virus and disease status. CASE PRESENTATION A novel adenovirus was identified in four captive bottlenose dolphins with self-limiting gastroenteritis. Viral detection and identification were achieved by: PCR-amplification from fecal samples; sequencing of partial adenovirus polymerase (pol) and hexon genes; producing the virus in HeLa cells, with PCR and immunofluorescence detection, and with sequencing of the amplified pol and hexon gene fragments. A causative role of this adenovirus for gastroenteritis was suggested by: 1) we failed to identify other potential etiological agents; 2) the exclusive detection of this novel adenovirus and of seropositivity for canine adenoviruses 1 and 2 in the four sick dolphins, but not in 10 healthy individuals of the same captive population; and 3) the virus disappeared from feces after clinical signs receded. The partial sequences of the amplified fragments of the pol and hexon genes were closest to those of adenoviruses identified in sea lions with fatal adenoviral hepatitis, and to a Genbank-deposited sequence obtained from a harbour porpoise. CONCLUSION These data suggest that adenovirus can cause self-limiting gastroenteritis in dolphins. This adenoviral infection can be detected by serology and by PCR detection in fecal material. Lack of signs of hepatitis in sick dolphins may reflect restricted tissue tropism or virulence of this adenovirus compared to those of the adenovirus identified in sea lions. Gene sequence-based phylogenetic analysis supports a common origin of adenoviruses that affect sea mammals. Our findings suggest the need for vigilance against adenoviruses in captive and wild dolphin populations

First molecular detection and characterization of herpesvirus and poxvirus in a Pacific walrus (Odobenus rosmarus divergens)

BACKGROUND Herpesvirus and poxvirus can infect a wide range of species: herpesvirus genetic material has been detected and amplified in five species of the superfamily Pinnipedia; poxvirus genetic material, in eight species of Pinnipedia. To date, however, genetic material of these viruses has not been detected in walrus (Odobenus rosmarus), another marine mammal of the Pinnipedia clade, even though anti-herpesvirus antibodies have been detected in these animals. CASE PRESENTATION In February 2013, a 9-year-old healthy captive female Pacific walrus died unexpectedly at L'Oceanografic (Valencia, Spain). Herpesvirus was detected in pharyngeal tonsil tissue by PCR. Phylogenetic analysis revealed that the virus belongs to the subfamily Gammaherpesvirinae. Poxvirus was also detected by PCR in skin, pre-scapular and tracheobronchial lymph nodes and tonsils. Gross lesions were not detected in any tissue, but histopathological analyses of pharyngeal tonsils and lymph nodes revealed remarkable lymphoid depletion and lymphocytolysis. Similar histopathological lesions have been previously described in bovine calves infected with an alphaherpesvirus, and in northern elephant seals infected with a gammaherpesvirus that is closely related to the herpesvirus found in this case. Intracytoplasmic eosinophilic inclusion bodies, consistent with poxviral infection, were also observed in the epithelium of the tonsilar mucosa. CONCLUSION To our knowledge, this is the first molecular identification of herpesvirus and poxvirus in a walrus. Neither virus was likely to have contributed directly to the death of our animal

Unusual striped dolphin mass mortality episode related to cetacean morbillivirus in the Spanish Mediterranean sea

BACKGROUND In the last 20 years, Cetacean Morbillivirus (CeMV) has been responsible for many die-offs in marine mammals worldwide, as clearly exemplified by the two dolphin morbillivirus (DMV) epizootics of 1990-1992 and 2006-2008, which affected Mediterranean striped dolphins (Stenella coeruleoalba). Between March and April 2011, the number of strandings on the Valencian Community coast (E Spain) increased. CASE PRESENTATION Necropsy and sample collection were performed in all stranded animals, with good state of conservation. Subsequently, histopathology, immunohistochemistry, conventional reverse transcription polymerase chain reaction (RT-PCR) and Universal Probe Library (UPL) RT-PCR assays were performed to identify Morbillivirus. Gross and microscopic findings compatible with CeMV were found in the majority of analyzed animals. Immunopositivity in the brain and UPL RT-PCR positivity in seven of the nine analyzed animals in at least two tissues confirmed CeMV systemic infection. Phylogenetic analysis, based on sequencing part of the phosphoprotein gene, showed that this isolate is a closely related dolphin morbillivirus (DMV) to that responsible for the 2006-2008 epizootics. CONCLUSION The combination of gross and histopathologic findings compatible with DMV with immunopositivity and molecular detection of DMV suggests that this DMV strain could cause this die-off event

Pilot Investigation of SARS-CoV-2 Variants in the Island of Sicily Prior to and in the Second Wave of the COVID-19 Pandemic

8 páginas, 2 tablas, 2 figuras. The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://www.ncbi.nlm.nih.gov/genbank/, OM510944; https://www.ncbi.nlm.nih.gov/genbank/, OM510945; https://www.ncbi. nlm.nih.gov/genbank/, OM510946; https://www.ncbi.nlm.nih. gov/genbank/, OM510947; https://www.ncbi.nlm.nih.gov/genbank/, OM510948; https://www.ncbi.nlm.nih.gov/genbank/, OM510949; https://www.ncbi.nlm.nih.gov/genbank/, OM51 0950; https://www.ncbi.nlm.nih.gov/genbank/, OM510951; and https://www.ncbi.nlm.nih.gov/genbank/, OM510952.After 2 years of the COVID-19 pandemic, we continue to face vital challenges stemming from SARS-CoV-2 variation, causing changes in disease transmission and severity, viral adaptation to animal hosts, and antibody/vaccine evasion. Since the monitoring, characterization, and cataloging of viral variants are important and the existing information on this was scant for Sicily, this pilot study explored viral variants circulation on this island before and in the growth phase of the second wave of COVID-19 (September and October 2020), and in the downslope of that wave (early December 2020) through sequence analysis of 54 SARS-CoV-2-positive samples. The samples were nasopharyngeal swabs collected from Sicilian residents by a state-run one-health surveillance laboratory in Palermo. Variant characterization was based on RT-PCR amplification and sequencing of four regions of the viral genome. The B.1.177 variant was the most prevalent one, strongly predominating before the second wave and also as the wave downsized, although its relative prevalence decreased as other viral variants, particularly B.1.160, contributed to virus circulation. The occurrence of the B.1.160 variant may have been driven by the spread of that variant in continental Europe and by the relaxation of travel restrictions in the summer of 2020. No novel variants were identified. As sequencing of the entire viral genome in Sicily for the period covered here was restricted to seven deposited viral genome sequences, our results shed some light on SARS-CoV-2 variant circulation during that wave in this insular region of Italy which combines its partial insular isolation with being a major entry point for the African immigration.This research received external funding to CR-G and EM from “Agencia Valenciana de Innovación: COVID-19, Ayudas de Concesión Directa a Soluciones Científico-Innovadoras Directamente Relacionadas Con La Lucha Contra La COVID-19,” (Ref COVID-19-203) and from “Conselleria de Innovación Universidades, Ciencia y Sociedad Digital: Subvenciones a Grupos de Investigación Emergentes” (Ref, GV/2021/163); to VR from the Agencia Estatal de Investigación of the Spanish Government (Ref PID2020-120322RB-C21) and from the European Commission–NextGeneration EU CSIC Global Health Platform, Spanish Ministry of Science and Innovation (Ref MCIN/AEI/10.13039/501100011033); and to the Istituto Zooprofilattico Sperimentale della Sicilia “A. Mirri” by the Project “COVID-19: Traiettorie Evolutive di SARS-CoV-2 ed Indagine Sul Ruolo Degli Animali” (IZS SI 03/20 RC), funded by the Italian Ministry of Health.Peer reviewe

A SARS-CoV-2 full genome sequence of the B.1.1 lineage sheds light on viral evolution in Sicily in late 2020

9 páginas, 2 figuras, 2 tablasTo investigate the influence of geographic constrains to mobility on SARS-CoV-2 circulation before the advent of vaccination, we recently characterized the occurrence in Sicily of viral lineages in the second pandemic wave (September to December 2020). Our data revealed wide prevalence of the then widespread through Europe B.1.177 variant, although some viral samples could not be classified with the limited Sanger sequencing tools used. A particularly interesting sample could not be fitted to a major variant then circulating in Europe and has been subjected here to full genome sequencing in an attempt to clarify its origin, lineage and relations with the seven full genome sequences deposited for that period in Sicily, hoping to provide clues on viral evolution. The obtained genome is unique (not present in databases). It hosts 20 single-base substitutions relative to the original Wuhan-Hu-1 sequence, 8 of them synonymous and the other 12 encoding 11 amino acid substitutions, all of them already reported one by one. They include four highly prevalent substitutions, NSP12:P323L, S:D614G, and N:R203K/G204R; the much less prevalent S:G181V, ORF3a:G49V and N:R209I changes; and the very rare mutations NSP3:L761I, NSP6:S106F, NSP8:S41F and NSP14:Y447H. GISAID labeled this genome as B.1.1 lineage, a lineage that appeared early on in the pandemic. Phylogenetic analysis also confirmed this lineage diagnosis. Comparison with the seven genome sequences deposited in late 2020 from Sicily revealed branching leading to B.1.177 in one branch and to Alpha in the other branch, and suggested a local origin for the S:G118V mutation.This research received external funding to CR-G from Conselleria de Innovación Universidades, Ciencia y Sociedad Digital: Subvenciones a Grupos de Investigación Emergentes (Ref, GV/2021/163); to VR from the Agencia Estatal de Investigación of the Spanish Government (Ref PID2020-120322RB-C21) and the European Commission-NextGeneration EU CSIC Global Health Platform, Spanish Ministry of Science and Innovation (Ref MCIN/AEI/10.13039/501100011033); and to the Istituto Zooprofilattico Sperimentale della Sicilia A. Mirri by the Project COVID-19: Traiettorie Evolutive di SARS-CoV-2 ed Indagine Sul Ruolo Degli Animali (IZS SI 03/20 RC), funded by the Italian Ministry of HealthPeer reviewe