EX VIVO STUDIES ON HOST RANGE AND TROPISM OF INFLUENZA A VIRUSES

Influenza A viruses (IAVs) are single stranded RNA viruses belonging to the Orthomyxoviridae family. These viruses exhibit high evolutionary rates and are present in a wide range of animal species. Host switching of IAVs is unpredictable and poses a great threat to human health. Therefore, understanding the underlying mechanisms driving such events is one of the current goals in influenza research. Among the different subtypes, H3N8 IAVs have shown a remarkable tendency to host species jumps. H3N8 equine influenza virus (EIV) is an avian-origin virus that circulated in horses for nearly 40 years before emerging in dogs in early 2000s. In the last decade H3N8 EIV has also been isolated from pigs, key hosts in influenza ecology, and other mammals. To understand the nature of host range determinants it is fundamental to study the dynamics of influenza pathogenesis at the site of infection. Respiratory explants constitute a suitable model to investigate virus replication in the target tissues of the host. This thesis aimed at thoroughly investigating the infection dynamics of host adapted and non-host adapted IAV infections in organ explants of pigs and horses. We first described the infection phenotype of host adapted viruses such as H3N2 swine influenza (SIV) and H3N8 equine influenza in tissues of their natural hosts to build a solid background knowledge on disease pathogenesis. Swine and equine explants were infected with SIV and EIV, respectively, and accurately monitored for viral replication and structural changes at the site of infection (chapters two and three). This extensive examination has added substantial information to previous literature and confirmed that swine and equine ex vivo systems support influenza replication and are suitable to study virus pathogenesis and at the same time observe the 3Rs ethos (Reduction, Replacement and Refinement). Next, we addressed our research interests on non-host adapted infections focusing on the H3N8 subtype. Enhancing the current knowledge on the role of evolution in the complex phenomenon of viral emergence is of utmost importance. To this end, in chapter four we compared the replication potential of phylogenetically distinct EIVs, each representing an evolutionary different period, in swine cell lines and respiratory explants. All tested EIVs replicated in cell lines whereas only the earliest EIV isolate (Uruguay/63) was able to infect swine explants with a marked tropism for the lower respiratory tract, demonstrating the presence of tissue-specific host barriers at the site of infection. The distinct phenotypes observed ex vivo support the view that evolutionary processes play important roles on host range and tropism of EIV. Nonetheless, when compared to SIV H3N2, EIV Uruguay/63 productively infected only a limited number of explants. H3N8 EIV has originated from the avian gene pool maintained in aquatic waterfowl. To further look into host range shifts of H3N8 IAVs, in chapter five we investigated the replication dynamics of H3N8 avian influenza viruses (AIVs) in equine tracheal explants. To give an ecologically plausible context to our experiments, we tested AIVs isolated from wild birds in Mongolia, a region densely populated with wild birds and horses. H3N8 AIVs infected tracheal explants, albeit to significantly lower levels and with a different infection phenotype compared to an EIV. This difference was evidenced as lower viral titres, absence of epithelial damage and difficult viral nucleoprotein detection in the infected tissue. These findings, coupled with serological evidence of AIV infections in horses in the field, suggest that introduction of viruses from the avian reservoir can occur and that further adaptive changes may be required for a successful establishment. Overall, by investigating the infection dynamics of IAVs in ex vivo cultures of the respiratory tract we have provided new insights into the different phenotypes displayed by host adapted and non-host adapted viruses at the site of infection. Our results support the hypothesis that viral evolution during long-term transmission of IAVs in host populations could result in dynamic changes in their host range. Such changes must be in line with ecological and epidemiological factors in order to allow the establishment of novel lineages in susceptible hosts. Finally, we have confirmed that organ explants can bridge important gaps between in vitro and in vivo experiments

EX VIVO STUDIES ON HOST RANGE AND TROPISM OF INFLUENZA A VIRUSES

Influenza A viruses (IAVs) are single stranded RNA viruses belonging to the Orthomyxoviridae family. These viruses exhibit high evolutionary rates and are present in a wide range of animal species. Host switching of IAVs is unpredictable and poses a great threat to human health. Therefore, understanding the underlying mechanisms driving such events is one of the current goals in influenza research. Among the different subtypes, H3N8 IAVs have shown a remarkable tendency to host species jumps. H3N8 equine influenza virus (EIV) is an avian-origin virus that circulated in horses for nearly 40 years before emerging in dogs in early 2000s. In the last decade H3N8 EIV has also been isolated from pigs, key hosts in influenza ecology, and other mammals. To understand the nature of host range determinants it is fundamental to study the dynamics of influenza pathogenesis at the site of infection. Respiratory explants constitute a suitable model to investigate virus replication in the target tissues of the host. This thesis aimed at thoroughly investigating the infection dynamics of host adapted and non-host adapted IAV infections in organ explants of pigs and horses. We first described the infection phenotype of host adapted viruses such as H3N2 swine influenza (SIV) and H3N8 equine influenza in tissues of their natural hosts to build a solid background knowledge on disease pathogenesis. Swine and equine explants were infected with SIV and EIV, respectively, and accurately monitored for viral replication and structural changes at the site of infection (chapters two and three). This extensive examination has added substantial information to previous literature and confirmed that swine and equine ex vivo systems support influenza replication and are suitable to study virus pathogenesis and at the same time observe the 3Rs ethos (Reduction, Replacement and Refinement). Next, we addressed our research interests on non-host adapted infections focusing on the H3N8 subtype. Enhancing the current knowledge on the role of evolution in the complex phenomenon of viral emergence is of utmost importance. To this end, in chapter four we compared the replication potential of phylogenetically distinct EIVs, each representing an evolutionary different period, in swine cell lines and respiratory explants. All tested EIVs replicated in cell lines whereas only the earliest EIV isolate (Uruguay/63) was able to infect swine explants with a marked tropism for the lower respiratory tract, demonstrating the presence of tissue-specific host barriers at the site of infection. The distinct phenotypes observed ex vivo support the view that evolutionary processes play important roles on host range and tropism of EIV. Nonetheless, when compared to SIV H3N2, EIV Uruguay/63 productively infected only a limited number of explants. H3N8 EIV has originated from the avian gene pool maintained in aquatic waterfowl. To further look into host range shifts of H3N8 IAVs, in chapter five we investigated the replication dynamics of H3N8 avian influenza viruses (AIVs) in equine tracheal explants. To give an ecologically plausible context to our experiments, we tested AIVs isolated from wild birds in Mongolia, a region densely populated with wild birds and horses. H3N8 AIVs infected tracheal explants, albeit to significantly lower levels and with a different infection phenotype compared to an EIV. This difference was evidenced as lower viral titres, absence of epithelial damage and difficult viral nucleoprotein detection in the infected tissue. These findings, coupled with serological evidence of AIV infections in horses in the field, suggest that introduction of viruses from the avian reservoir can occur and that further adaptive changes may be required for a successful establishment. Overall, by investigating the infection dynamics of IAVs in ex vivo cultures of the respiratory tract we have provided new insights into the different phenotypes displayed by host adapted and non-host adapted viruses at the site of infection. Our results support the hypothesis that viral evolution during long-term transmission of IAVs in host populations could result in dynamic changes in their host range. Such changes must be in line with ecological and epidemiological factors in order to allow the establishment of novel lineages in susceptible hosts. Finally, we have confirmed that organ explants can bridge important gaps between in vitro and in vivo experiments.I virus dell’Influenza A sono virus ad RNA con genoma segmentato appartenenti alla famiglia Orthomyxoviridae la cui ecologia si distingue per l’ampio spettro di ospiti ed elevato tasso evolutivo. Le frequenti trasmissioni inter-specie ad oggi risultano impredevibili e rappresentano un elevato rischio per la salute umana. Per questo motivo il fronte della ricerca sui virus influenzali è concentrato sullo studio della trasmissione dell’influenza da un ospite all’altro. Tra i diversi sottotipi esistenti, il sottotipo H3N8 ha dimostrato una notevole tendenza al salto di specie. L’influenza equina H3N8 (EIV) è un virus di origine aviare che ha ampiamente circolato nei cavalli per circa 40 anni prima di saltare la barriera di specie e stabilirsi nella popolazione canina dando origine all’influenza canina all’inizio degli anni 2000. Recentemente l’influenza equina è stata occasionalmente isolata anche da maiali, ospiti molto importanti nell’ecologia dell’influenza in quanto suscettibili a numerosi virus di diversa origine animale, e da altri mammiferi. Per studiare a fondo il tropismo dei virus influenzali nei diversi ospiti è fondamentale osservarne il comportamento nei tessuti target. Gli espianti d’organo rappresentano un’alternativa alle sperimentazioni in vivo, permettendo di caratterizzare il potenziale replicativo dei virus e riducendo il numero di animali utilizzati a fini sperimentali. Lo scopo di questa tesi è stato caratterizzare infezioni specie-specifiche e non specie-specifiche causate da virus influenzali di tipo A in espianti d’organo di maiale e cavallo. In primo luogo, con la finalità di acquisire un’importante conoscenza di base sulla patogenesi della malattia, abbiamo caratterizzato due infezioni specie-specifiche nei tessuti target delle rispettive specie ospite: l’influenza suina H3N2 e l’influenza equina H3N8 (capitoli secondo e terzo, rispettivamente). In seguito all’infezione, per ciascun virus è stato descritto un fenotipo d’infezione basato sulla crescita virale e sui cambi strutturali nei tessuti interessati. I risultati ottenuti hanno aggiunto importanti informazioni alla letteratura preesistente ed hanno confermato la sensibilità di questa metodica ex vivo per lo studio della patogenesi di virus influenzali nel pieno rispetto del principio delle 3R (Reduction, Replacement and Refinement). Successivamente abbiamo utilizzato gli espianti d’organo per approfondire lo studio delle infezioni non specie-specifiche causate dal sottotipo H3N8. Lo scopo di questo lavoro è stato valutare l’impatto dell’evoluzione naturale di un virus (dovuta all’ampia circolazione) sul suo spettro d’ospite. Virus dell’influenza equina, filogeneticamente distinti e rappresentanti periodi evolutivi diversi, sono stati testati su linee cellulari primarie di maiale ed espianti d’organo suini (capitolo quarto). Tutti i virus inclusi nello studio sono stati in grado di replicare nelle linee cellulari mentre in espianti d’organo soltanto il primo isolato di influenza equina (Uruguay/63) è stato in grado di infettare diversi tessuti, mostrando un particolare tropismo per le vie respiratorie profonde. Questi dati dimostrano in primis la presenza di barriere specifiche a livello dei tessuti dell’ospite. Inoltre, le differenze osservate tra virus appartenenti a periodi evolutivi diversi suggeriscono che l’evoluzione ha un ruolo chiave sul tropismo e sullo spettro d’ospite dei virus dell’influenza equina. Tuttavia, pur replicando in tessuti suini, il virus Uruguay/63 ha infettato un numero minore di espianti rispetto ad un virus specie-specifico utilizzato come controllo positivo. Il virus dell’influenza equina H3N8 è originato da un virus aviare mantenuto nel serbatoio rappresentato dai volatili selvatici. Con la finalità di approfondire ulteriormente le nostre conoscenze sul sottotipo H3N8 ed il suo spettro d’ospite, nel quinto capitolo abbiamo valutato il potenziale replicativo di virus dell’influenza aviare in espianti di trachea di cavallo. Per dare un contesto verosimile ai nostri esperimenti, abbiamo utilizzato virus isolati da volatili selvatici in Mongolia, regione densamente popolata da volatili selvatici e cavalli. I virus testati sono stati in grado di replicare negli espianti di trachea di cavallo mostrando però titoli significativamente più bassi rispetto ad un virus specie-specifico utilizzato come controllo positivo. Inoltre, al contrario di quanto osservato con il virus equino, i virus aviari non hanno causato nessun danno epiteliale e la nucleoproteina virale è stata difficilmente identificata nei tessuti colpiti. L’evidenza sierologica di cavalli infetti con virus aviari in campo supporta i nostri risultati e la teoria per cui contatti sporadici con questi virus avvengono ma affinché siano in grado di stabilirsi nella popolazione equina è necessario un ulteriore adattamento. In conclusione, infettando espianti d’organo con virus specie-specifici e non, abbiamo fornito nuova evidenza di importanti differenze fenotipiche nei tessuti target. Inoltre, abbiamo dimostrato come l’evoluzione di un virus ne possa influenzare il tessuto tropismo e di conseguenza lo spettro d’ospite. In fine, abbiamo sottolineato come l’utilizzo di una metodica ex vivo possa colmare differenze fondamentali tra metodiche in vitro ed in vivo

Influenza A viruses grow in human pancreatic cells and cause pancreatitis and diabetes in an animal model.

Influenza A viruses commonly cause pancreatitis in naturally and experimentally infected animals. In this study we report the results of in vivo investigations carried out to establish whether influenza infection could cause metabolic disorders linked to pancreatic infection. In addition, in vitro tests in human pancreatic islets and in human pancreatic cell lines were performed to evaluate viral growth and cell damage.Infection of an avian model with two low pathogenicity avian influenza isolates caused pancreatic damage resulting in hyperlipasemia in over 50% of subjects, which evolved into hyperglycemia and subsequently diabetes. Histopathology of the pancreas showed signs of an acute infection resulting in severe fibrosis and disruption of the structure of the organ. Influenza nucleoprotein was detected by IHC in the acinar tissue.Human seasonal H1N1 and H3N2 viruses and avian H7N1 and H7N3 influenza isolates were able to infect a selection of human pancreatic cell lines. Human viruses were also shown to be able to infect human pancreatic islets. In situ hybridization assays indicated that viral nucleoprotein could be detected in beta cells. The cytokine activation profile indicated a significant increase of MIG/CXCL9, IP-10/CXCL10, RANTES/CCL5, MIP1b/CCL4, Groa/CXCL1, IL8/CXCL8, TNFa and IL-6.Our findings indicate that influenza infection may play a role as causative agent of pancreatitis and diabetes in humans and other mammals

Persistence and clearance of Ebola virus RNA from seminal fluid of Ebola virus disease survivors: a longitudinal analysis and modelling study

Background: By January, 2016, all known transmission chains of the Ebola virus disease (EVD) outbreak in west Africa had been stopped. However, there is concern about persistence of Ebola virus in the reproductive tract of men who have survived EVD. We aimed to use biostatistical modelling to describe the dynamics of Ebola virus RNA load in seminal fluid, including clearance parameters.Methods: In this longitudinal study, we recruited men who had been discharged from three Ebola treatment units in Guinea between January and July, 2015. Participants provided samples of seminal fluid at follow-up every 3–6 weeks, which we tested for Ebola virus RNA using quantitative real-time RT-PCR. Representative specimens from eight participants were then inoculated into immunodeficient mice to test for infectivity. We used a linear mixed-effect model to analyse the dynamics of virus persistence in seminal fluid over time.Findings: We enrolled 26 participants and tested 130 seminal fluid specimens; median follow up was 197 days (IQR 187–209 days) after enrolment, which corresponded to 255 days (228–287) after disease onset. Ebola virus RNA was detected in 86 semen specimens from 19 (73%) participants. Median duration of Ebola virus RNA detection was 158 days after onset (73–181; maximum 407 days at end of follow-up). Mathematical modelling of the quantitative time-series data showed a mean clearance rate of Ebola virus RNA from seminal fluid of −0·58 log units per month, although the clearance kinetic varied greatly between participants. Using our biostatistical model, we predict that 50% and 90% of male survivors clear Ebola virus RNA from seminal fluid at 115 days (90% prediction interval 72–160) and 294 days (212–399) after disease onset, respectively. We also predicted that the number of men positive for Ebola virus RNA in affected countries would decrease from about 50 in January 2016, to fewer than 1 person by July, 2016. Infectious virus was detected in 15 of 26 (58%) specimens tested in mice.Interpretation: Time to clearance of Ebola virus RNA from seminal fluid varies greatly between individuals and could be more than 13 months. Our predictions will assist in decision-making about surveillance and preventive measures in EVD outbreaks.Funding: This study was funded by European Union's Horizon 2020 research and innovation programme, Directorate-General for International Cooperation and Development of the European Commission, Institut national de la santé et de la recherche médicale (INSERM), German Research Foundation (DFG), and Innovative Medicines Initiative 2 Joint Undertaking.</br