24 research outputs found
Revealing the role of early immune responses to control MERS-CoV infection in a camelid model
No full textEl coronavirus de la síndrome respiratòria de l’Orient Mitjà (en anglès, MERS-CoV) causa una elevada letalitat a les persones. Es pensa que aquest patogen zoonòtic emergent ha evolucionat a partir d’un coronavirus de rat penat, però el dromedari és l’única font confirmada d’infecció zoonòtic. Malgrat la reducció global de casos de MERS des de 2015, el virus segueix essent enzoòtic en dromedaris de la Península Aràbiga i Àfrica.
A les persones, els casos greus associats a la infecció per MERS-CoV són la conseqüència d’un dany alveolar difús. Pel contrari, els camèlids, el principal reservori del virus, són portadors asimptomàtics del MERS-CoV, fet que suggereix un paper fonamental de la resposta immunitària innata en el control de la infecció en aquests animals.
L’objectiu de l’estudi I d’aquesta tesi doctoral avaluà la transcripció de gens involucrats en la resposta immunitària en el tracte respiratori d’alpaques infectades amb MERS-CoV Qatar15/2015 (clade B). Els IFN tipus I i III es van transcriure al seu màxim nivell només a la mucosa nasal de les alpaques i en concomitància amb el pic de la infecció (moment de màxima càrrega vírica al tracte respiratori), fet que es va donar 2 dies després de la infecció (dpi). Aquesta transcripció es va associar a la inducció de gens estimulats per interferó (ISG) al llarg de tot el tracte respiratori. Simultàniament a la infiltració focal lleu de leucòcits a la mucosa i submucosa nasal, també es va observar una regulació positiva de la citocina antiinflamatòria IL10 i una transcripció esmorteïda de gens proinflamatoris sota el control de NF-κB. En el pulmó, la transcripció primerenca (1 dpi) de quimiocines (CCL2 i CCL3) es va correlacionar amb una acumulació lleu i transitòria de leucòcits principalment mononuclears. Una regulació estricta dels IFNs en pulmons amb expressió de ISGs i una resposta inflamatòria controlades podrien contribuir a l’eliminació del virus sense causar dany tissular. Per tant, la mucosa nasal, la principal diana del MERS-CoV en camèlids, és fonamental per impulsar una resposta immunitària innata eficient basada en l’activació d’ISGs, així com dels efectes antiinflamatoris duals dels IFNs de tipus III i l’IL10.
Tots els virus epidèmics contemporanis pertanyen al clade B. Per tant, les soques d’aquest clade B sembla que tenen avantatges adaptatives sobre el clade A en persones i hostes reservoris. A l’estudi II d’aquesta tesi doctoral es va comparar una soca de clade A de les epidèmies inicials (EMC / 2012) amb una soca de la clade B (Jordan-1/2015) en un model d’alpaca que avaluà paràmetres virològics i immunològics. A més, la soca Jordan-1/2015 té una deleció parcial d’aminoàcids (aa) en el motif d’unió de l’ARN bicatenari (ds) de la proteïna del marc de lectura oberta 4a (ORF4a). Els animals inoculats amb la soca Jordan-1/2015 van tenir una major replicació del MERS-CoV al tracte respiratori i una major disseminació viral nasal, fet que indicaria una millor aptitud i capacitat de transmissió que la seva contrapart de la soca del clade A. A la mucosa nasal, la soca Jordan-1/2015 va provocar una resposta primerenca d’IFNs a 1 dpi, cosa que confirmaria el paper de l’ORF4a com antagonista d’IFNs en condicions in vivo. No obstant, les dues soques van provocar la màxima transcripció d’ISGs durant el pic de la infecció (2 dpi), correlacionant-se amb una disminució de les càrregues víriques tissulars (evident a 3 dpi). L’anàlisi d’alineació del genoma va mostrar diverses substitucions d’aa específiques del clade B que es produeixen a la replicasa i la proteïna S, fet que podria explicar una millor adaptació de les soques del clade B en els camèlids.El coronavirus del síndrome respiratorio de Oriente Medio (del inglés, MERS-CoV) causa una alta letalidad en los seres humanos. Se cree que éste patógeno ha evolucionado a partir de un coronavirus de murciélago, pero el dromedario es la única fuente confirmada de infección zoonótica. A pesar de la reducción global de casos de MERS desde 2015, el virus sigue siendo enzoótico en dromedarios en la Península Arábiga y África.
En los seres humanos, los casos graves asociados a la infección por MERS-CoV son la consecuencia de un daño alveolar difuso. Por el contrario, los camélidos, el principal reservorio del virus, son portadores asintomáticos del MERS-CoV, lo que sugiere un papel crucial de la respuesta inmunitaria innata en el control de la infección en estos animales.
El objetivo del estudio I de esta tesis doctoral fue evaluar la transcripción de genes involucrados en la respuesta inmune en el tracto respiratorio de alpacas infectadas con MERS-CoV Qatar15/2015 (clado B). Los IFN tipo I y III se transcribieron a su máximo nivel sólo en la mucosa nasal de las alpacas y en concomitancia con el pico de infección (momento de mayor carga vírica en el tracto respiratorio), que ocurrió 2 días después de la inoculación (dpi). Esta transcripción se asoció a la inducción de genes estimulados por interferón (ISG) a lo largo de todo el tracto respiratorio. Simultáneamente a la infiltración focal leve de leucocitos en la mucosa nasal y submucosa, también se observó una regulación positiva de la citocina antiinflamatoria IL10 y una transcripción amortiguada de genes proinflamatorios bajo control de NF-κB. En el pulmón, la transcripción temprana (1 dpi) de quimiocinas (CCL2 y CCL3) se correlacionó con una acumulación leve y transitoria de leucocitos principalmente mononucleares. Una regulación estricta de los IFNs en los pulmones con expresión de ISG y una respuesta inflamatoria controladas podría contribuir a la eliminación del virus sin causar daño tisular. Por tanto, la mucosa nasal, la principal diana del MERS-CoV en los camélidos, es fundamental para impulsar una respuesta inmune innata eficaz basada en la activación de ISG, así como en los efectos antiinflamatorios duales de los IFNs de tipo III y la IL10.
Todos los virus epidémicos contemporáneos pertenecen al clado B. Por lo tanto, las cepas de MERS-CoV del clado B parecen mostrar ventajas adaptativas sobre el clado A en humanos / huéspedes reservorios. En el estudio II de esta tesis doctoral se comparó una cepa de clado A epidémico temprano (EMC / 2012) con una cepa de clado B (Jordan-1/2015) en un modelo de alpaca que evaluó parámetros virológicos e inmunológicos. Además, la cepa Jordan-1/2015 tiene una deleción parcial de aminoácidos (aa) en el motivo de unión del ARN bicatenario (ds) de la proteína del marco de lectura abierto 4a (ORF4a). Los animales inoculados con la cepa Jordan-1/2015 tuvieron una mayor replicación de MERS-CoV en el tracto respiratorio y una mayor diseminación viral nasal, lo que indica una aparente mejor aptitud y capacidad de transmisión que su contraparte de la cepa del clado A. En la mucosa nasal, la cepa Jordan-1/2015 provocó una respuesta de IFNs temprana a 1 dpi, lo que confirma el papel de ORF4a como antagonista de IFNs in vivo. Sin embargo, ambas cepas provocaron la máxima transcripción de ISGs en el pico de la infección (a 2 dpi), correlacionándose con una disminución de las cargas virales tisulares (evidente a 3 dpi). La alineación del genoma reveló varias sustituciones de aa específicas del clado B que se producen en la replicasa y la proteína S; ello podría explicar una mejor adaptación de las cepas del clado B en los camélidos.Middle East respiratory syndrome coronavirus (MERS-CoV) is the etiological agent of a respiratory disease able to cause high mortality in humans. Severe cases associated to MERS-CoV infection are the consequence of the diffuse alveolar damage triggered by the pro-inflammatory cytokine storm and impaired interferon (IFN) responses. By contrast, camelids, the main virus reservoir, are asymptomatic MERS-CoV carriers, suggesting a crucial role for innate immune responses in controlling the infection. In study I of this dissertation, we aim to demonstrate this hypothesis by monitoring the transcription of immune response genes in the respiratory tract of MERS-CoV Qatar15/2015 (clade B strain) infected alpacas. Concomitant to the peak of infection, occurring at 2 days post inoculation (dpi), type I and III IFNs were maximally transcribed only in the nasal mucosa of alpacas, provoking the induction of interferon stimulated genes (ISGs) along the whole respiratory tract. Simultaneous to mild focal infiltration of leukocytes in nasal mucosa and submucosa, upregulation of the anti-inflammatory cytokine IL10 and dampened transcription of pro-inflammatory genes under NF-κB control were observed. In the lung, early (1 dpi) transcription of chemokines (CCL2 and CCL3) correlated with a transient accumulation of mainly mononuclear leukocytes. A tight regulation of IFNs in lungs with expression of ISGs and controlled inflammatory responses, might contribute to virus clearance without causing tissue damage. Thus, the nasal mucosa, the main target of MERS-CoV in camelids, is central in driving an efficient innate immune response based on triggering ISGs as well as the dual anti-inflammatory effects of type III IFNs and IL10.
While MERS-CoV strains from the Middle East region are subdivided into two clades (A and B), all the contemporary epidemic viruses belong to clade B. Thus, clade B MERS-CoV strains must display adaptive advantages over clade A in humans/reservoir hosts. Therefore, in study II of this dissertation, we compared an early epidemic clade A strain (EMC/2012) with a clade B strain (Jordan-1/2015) in an alpaca model monitoring virological and immunological parameters. Further, the Jordan-1/2015 strain has a partial amino acid (aa) deletion in the double stranded (ds) RNA binding motif of the open reading frame ORF 4a protein. Animals inoculated with the Jordan-1/2015 strain had higher MERS-CoV replicative capacities in the respiratory tract and larger nasal viral shedding, indicating a better fitness and transmission capability than its clade A strain counterpart. In the nasal mucosa, the Jordan-1/2015 strain provoked an early IFN response on 1-day post inoculation (dpi), confirming the role of ORF4a as an IFN antagonist in vivo. However, both strains provoked at the peak of infection (on 2 dpi) maximal transcription of ISGs correlating with decreased tissular viral loads observed on 3 dpi. Genome alignment analysis revealed several clade B specific aa substitutions occurring in the replicase and the S protein which could explain a better adaptation of clade B strains in camelid hosts.
Overall, the results exposed in the present thesis highlight the complex interactions between MERS-CoV and host factors through which camelids control the infection in a short period of time. In addition, MERS-CoV strains are still evolving acquiring enhanced replicative fitness as shown in alpacas in the present study. This is reflected in the field by the dominance of MERS-CoV clade B strains over early epidemic clade A strains in humans and camelids.Universitat Autònoma de Barcelona. Programa de Doctorat en Medicina i Sanitat Animal
Flow Resistance Characteristics of a Specific Fuel RP-3 in Helical Tubes at Supercritical Pressure With Uniform Heat Flux
No full textThe flow resistance characteristics of aviation kerosene RP-3 in horizontal helical tubes at the supercritical pressure under heating condition are investigated. Both pressure drop and friction factor were examined under uniform heat flux of 50kW/m2−300kW/m2, mass flux from 786kg/m2s to 1375kg/m2s, and helical diameter from 20mm to 40mm. The influence of viscous factors on the resistance is analyzed to explore flow characteristics in a helical tube and provide a reference for the design of heat exchangers.
Friction factor decreases with the increase of heat flux at low inlet temperatures 323K and 423K. It is explained that the viscosity changes more dramatically than the density. When the fluid inlet temperature is 523K and the fluid mean temperature Tb is close to pseudo-critical temperature, frictional flow resistance becomes significantly larger Tpc due to huge variations in thermal properties in the radical direction. The effect of centrifugal force makes the friction factor decline slowly. The friction factor goes up with the enlargement of mass flux when Tb>0.81Tpc. This phenomenon is caused by the larger radial velocity gradient under the large mass flux. Different helical diameters play the leading roles for the bending flow in the tubes.</jats:p
Enhanced antiviral immunity and dampened inflammation in llama lymph nodes upon MERS-CoV sensing : bridging innate and adaptive cellular immune responses in camelid reservoirs
No full textMiddle East respiratory syndrome coronavirus (MERS-CoV) infection can cause fatal pulmonary inflammatory disease in humans. Contrarily, camelids and bats are the main reservoir hosts, tolerant for MERS-CoV replication without suffering clinical disease. Here, we isolated cervical lymph node (LN) cells from MERS-CoV convalescent llamas and pulsed them with two different viral strains (clades B and C). Viral replication was not supported in LN, but a cellular immune response was mounted. Reminiscent Th1 responses (IFN-γ, IL-2, IL-12) were elicited upon MERS-CoV sensing, accompanied by a marked and transient peak of antiviral responses (type I IFNs, IFN-λ3, ISGs, PRRs and TFs). Importantly, expression of inflammatory cytokines (TNF-α, IL-1β, IL-6, IL-8) or inflammasome components (NLRP3, CASP1, PYCARD) was dampened. The role of IFN-λ3 to counterbalance inflammatory processes and bridge innate and adaptive immune responses in camelid species is discussed. Our findings shed light into key mechanisms on how reservoir species control MERS-CoV in the absence of clinical disease
Enhanced antiviral immunity and dampened inflammation in llama lymph nodes upon MERS-CoV sensing: bridging innate and adaptive cellular immune responses in camelid reservoirs
No full textMiddle East respiratory syndrome coronavirus (MERS-CoV) infection can cause fatal pulmonary inflammatory disease in humans. Contrarily, camelids and bats are the main reservoir hosts, tolerant for MERS-CoV replication without suffering clinical disease. Here, we isolated cervical lymph node (LN) cells from MERS-CoV convalescent llamas and pulsed them with two different viral strains (clades B and C). Viral replication was not supported in LN, but a cellular immune response was mounted. Reminiscent Th1 responses (IFN-γ, IL-2, IL-12) were elicited upon MERS-CoV sensing, accompanied by a marked and transient peak of antiviral responses (type I IFNs, IFN-λ3, ISGs, PRRs and TFs). Importantly, expression of inflammatory cytokines (TNF-α, IL-1β, IL-6, IL-8) or inflammasome components (NLRP3, CASP1, PYCARD) was dampened. The role of IFN-λ3 to counterbalance inflammatory processes and bridge innate and adaptive immune responses in camelid species is discussed. Our findings shed light into key mechanisms on how reservoir species control MERS-CoV in the absence of clinical disease
Enhanced antiviral immunity and dampened inflammation in llama lymph nodes upon MERS-CoV sensing: bridging innate and adaptive cellular immune responses in camelid reservoirs
No full textMiddle East respiratory syndrome coronavirus (MERS-CoV) infection can cause fatal pulmonary inflammatory disease in humans. Contrarily, camelids and bats are the main reservoir hosts, tolerant for MERS-CoV replication without suffering clinical disease. Here, we isolated cervical lymph node (LN) cells from MERS-CoV convalescent llamas and pulsed them with two different viral strains (clades B and C). Viral replication was not supported in LN, but a cellular immune response was mounted. Reminiscent Th1 responses (IFN-γ, IL-2, IL-12) were elicited upon MERS-CoV sensing, accompanied by a marked and transient peak of antiviral responses (type I IFNs, IFN-λ3, ISGs, PRRs and TFs). Importantly, expression of inflammatory cytokines (TNF-α, IL-1β, IL-6, IL-8) or inflammasome components (NLRP3, CASP1, PYCARD) was dampened. The role of IFN-λ3 to counterbalance inflammatory processes and bridge innate and adaptive immune responses in camelid species is discussed. Our findings shed light into key mechanisms on how reservoir species control MERS-CoV in the absence of clinical disease.This study was performed as part of the Zoonotic Anticipation and Preparedness Initiative (ZAPI) [Innovative Medicines initiative (IMI) grant 115760] and the Veterinary Biocontained research facility Network (VetBioNet) (EU Grant Agreement INFRA-2016-1 N°731014) projects, with assistance and financial support from IMI and the European Commission and contributions from EFPIA partners. JR was partially supported by the VetBioNet. IRTA is supported by CERCA Programme/Generalitat de Catalunya.info:eu-repo/semantics/publishedVersio
Middle East respiratory coronavirus (MERS-CoV) internalized by llama alveolar macrophages does not result in virus replication or induction of pro-inflammatory cytokines
No full textSevere Middle East respiratory syndrome (MERS) is characterized by massive infiltration of immune cells
in lungs. MERS-coronavirus (MERS-CoV) replicates in vitro in human macrophages, inducing high proinflammatory responses. In contrast, camelids, the main reservoir for MERS-CoV, are asymptomatic
carriers. Although limited infiltration of leukocytes has been observed in the lower respiratory tract of
camelids, their role during infection remains unknown. Here we studied whether llama alveolar macrophages (LAMs) are susceptible to MERS-CoV infection and can elicit pro-inflammatory responses.
MERS-CoV did not replicate in LAMs; however, they effectively capture and degrade viral particles.
Moreover, transcriptomic analyses showed that LAMs do not induce pro-inflammatory cytokines upon
MERS-CoV sensing.This study was performed as part of the Zoonotic Anticipation and Preparedness Initiative (ZAPI project) [Innovative Medicines initiative (IMI) grant 115760] and the VetBioNet project (EU Grant Agreement INFRA-2016-1 Nº731014), with assistance and financial support from IMI and the European Commission and contributions from EFPIA partners and from grant RTI2018-09445-B-100 from the Ministry of Science and Innovation of Spain (to C.R.). J.R. was partially supported by the VetBioNet project. IRTA is supported by CERCA Programme/Generalitat de Catalunya.info:eu-repo/semantics/publishedVersio
Desenvolupen un nou model per estudiar la infecció del MERS-coronavirus al laboratori
No full textInvestigadors de la Unitat Mixta IRTA-UAB en Salut Animal han desenvolupat i validat un tècnica de cultiu de teixit respiratori de llama que permet estudiar per què els camèlids superen la infecció causada pel MERS-CoV sense desenvolupar signes clínics. Aquesta metodologia permetrà testar productes terapèutics per als humans front a aquest coronavirus, reduint així l'ús d'animals amb finalitats experimentals.Investigadores de la Unidad Mixta IRTA-UAB en Salud Animal han desarrollado y validado un técnica de cultivo de tejido respiratorio de llama que permite estudiar por qué los camélidos superan la infección causada por el MERS-CoV sin desarrollar signos clínicos. La metodología permitirá testar productos terapéuticos para humanos frente a este coronavirus, reduciendo así el uso de animales con fines experimentales.Researchers at the IRTA-UAB Joint Research Unit in Animal Health have developed and validated a technique for the culture of llama respiratory tissue that permits to study why camelids overcome infection caused by MERS-CoV without developing clinical signs. The methodology will allow to test therapeutic products for humans against this coronavirus, thus reducing the use of animals for experimental purposes
Enhanced replication fitness of MERS-CoV clade B over clade A strains in camelids explains the dominance of clade B strains in the Arabian Peninsula
No full textMiddle East respiratory syndrome coronavirus (MERS-CoV) continues infecting humans and dromedary camels. While MERS-CoV strains from the Middle East region are subdivided into two clades (A and B), all the contemporary epidemic viruses belong to clade B. Thus, MERS-CoV clade B strains may display adaptive advantages over clade A in humans and/or reservoir hosts. To test this hypothesis in vivo, we compared an early epidemic clade A strain (EMC/2012) with a clade B strain (Jordan-1/2015) in an alpaca model monitoring virological and immunological parameters. Further, the Jordan-1/2015 strain has a partial amino acid (aa) deletion in the double-stranded (ds) RNA binding motif of the open reading frame ORF4a protein. Animals inoculated with the Jordan-1/2015 variant had higher MERS-CoV replicative capabilities in the respiratory tract and larger nasal viral shedding. In the nasal mucosa, the Jordan-1/2015 strain caused an early IFN response, suggesting a role for ORF4a as a moderate IFN antagonist in vivo. However, both strains elicited maximal transcription of antiviral interferon-stimulated genes (ISGs) at the peak of infection on 2 days post inoculation, correlating with subsequent decreases in tissular viral loads. Genome alignment analysis revealed several clade B-specific amino acid substitutions occurring in the replicase and the S proteins, which could explain a better adaptation of clade B strains in camelid hosts. Differences in replication and shedding reported herein indicate a better fitness and transmission capability of MERS-CoV clade B strains than their clade A counterparts.info:eu-repo/semantics/publishedVersio
Type I and III IFNs produced by the nasal epithelia and dimmed inflammation are features of alpacas resolving MERS-CoV infection.
No full textWhile MERS-CoV (Middle East respiratory syndrome Coronavirus) provokes a lethal disease in humans, camelids, the main virus reservoir, are asymptomatic carriers, suggesting a crucial role for innate immune responses in controlling the infection. Experimentally infected camelids clear infectious virus within one week and mount an effective adaptive immune response. Here, transcription of immune response genes was monitored in the respiratory tract of MERS-CoV infected alpacas. Concomitant to the peak of infection, occurring at 2 days post inoculation (dpi), type I and III interferons (IFNs) were maximally transcribed only in the nasal mucosa of alpacas, while interferon stimulated genes (ISGs) were induced along the whole respiratory tract. Simultaneous to mild focal infiltration of leukocytes in nasal mucosa and submucosa, upregulation of the anti-inflammatory cytokine IL10 and dampened transcription of pro-inflammatory genes under NF-κB control were observed. In the lung, early (1 dpi) transcription of chemokines (CCL2 and CCL3) correlated with a transient accumulation of mainly mononuclear leukocytes. A tight regulation of IFNs in lungs with expression of ISGs and controlled inflammatory responses, might contribute to virus clearance without causing tissue damage. Thus, the nasal mucosa, the main target of MERS-CoV in camelids, seems central in driving an efficient innate immune response based on triggering ISGs as well as the dual anti-inflammatory effects of type III IFNs and IL10
Middle East respiratory coronavirus (MERS-CoV) internalized by llama alveolar macrophages does not result in virus replication or induction of pro-inflammatory cytokines
No full textSevere Middle East respiratory syndrome (MERS) is characterized by massive infiltration of immune cells in lungs. MERS-coronavirus (MERS-CoV) replicates in vitro in human macrophages, inducing high pro-inflammatory responses. In contrast, camelids, the main reservoir for MERS-CoV, are asymptomatic carriers. Although limited infiltration of leukocytes has been observed in the lower respiratory tract of camelids, their role during infection remains unknown. Here we studied whether llama alveolar macrophages (LAMs) are susceptible to MERS-CoV infection and can elicit pro-inflammatory responses. MERS-CoV did not replicate in LAMs; however, they effectively capture and degrade viral particles. Moreover, transcriptomic analyses showed that LAMs do not induce pro-inflammatory cytokines upon MERS-CoV sensing.This study was performed as part of the Zoonotic Anticipation and Preparedness Initiative (ZAPI project) [Innovative Medicines initiative (IMI) grant 115760] and the VetBioNet project (EU Grant Agreement INFRA-2016-1 Nº731014), with assistance and financial support from IMI and the European Commission and contributions from EFPIA partners and from grant RTI2018-09445-B-100 from the Ministry of Science and Innovation of Spain (to C.R.). J.R. was partially supported by the VetBioNet project. IRTA is supported by CERCA Programme/Generalitat de Catalunya.S
