Mechanism Of Exosomes Regulating Newcastle Disease Virus Infection In Vivo

Newcastle disease virus(NDV)is a single stranded negative strand RNA virus.It mainly infects poultry and causes respiratory and digestive diseases in susceptible animals.Pathological changes such as epithelial cell degeneration,necrosis and interstitial pneumonia can occur in infected animals,and airway blockage and dyspnea can occur in severe cases.NDV has the ability of genetic variation,can produce transboundary infection between different hosts,has strong infectivity,causes high group mortality of infected animals,and causes huge losses to Chinese aquaculture and international trade.Extracellular vesicles(Ex)are extracellular membrane structures that can carry bioactive molecules in cells,such as proteins,nucleic acids and metabolites,and carry out information transmission and regulation between cells.Exosomes play an important role in pathogen infection and immune response.Studies have shown that exosomes can promote infection of many pathogens by releasing biomolecules,and can also deliver viral RNA and proteins to facilitate their replication and spread in host cells.Our team’s previous research results showed that in vitro infection experiments,NDV infection promoted the production of exosomes,and these exosomes further promoted viral infection,which enabled NDV to rapidly proliferate between cells.Whether exosomes are involved in the infection and pathogenesis of viruses in animals has been a hot topic in domestic and foreign researches,which has important theoretical significance for the in-depth interpretation of the pathogenesis of NDVTherefore,this study aims to explore the influence of exosomes on Newcastle disease virus infection in vivo and its mechanism.The main research contents are as follows:1.Study on effect of NDV Ex on NDV infection in vivoIn this study,PEG purification method was used to isolate CEF-derived NDV Ex(i.e.exosomes secreted by NDV-infected cells).Viral load and pathological changes of chicken tissue after infection with NDV and NDV+NDV Ex were studied by qPCR and HE stained tissue sections.The results showed that NDV Ex could promote NDV replication in the lung,spleen and liver,enhance the persistence of infection,and lead to more serious inflammatory cell infiltration,alveolar damage and enlargement of alveolar cavity and other pathological changes in the lung,and more serious inflammatory cell infiltration,follicular center inflammation and tissue damage in the spleen.The liver showed more serious inflammatory cell infiltration,hepatic cord disorder and other pathological changes.2.Effect of NDV Ex on polarizing typing of chicken alveolar macrophagesDiR is a near infrared fluorescent dye that can be used to label biological samples.DiR labeled NDV Ex was injected into chickens from the subwing vein,and NDV Ex was traced in vivo by small animal imager.The results showed that NDV Ex could be stably labeled and tracked in chickens,and was organoselective to the lung,suggesting that exosomes may mainly act on the lung to regulate NDV infection in vivo.Alveolar macrophages in the lung were isolated,and the internalization of NDV Ex by alveolar macrophages and the mRNA transcription level of immune-related factors were detected by IFA and qPCR.The results showed that NDV Ex could be phagocytic by alveolar macrophages.In addition,NDV Ex can significantly increase the M1-type polarization factor(IL-1β,iNOS),M2-type polarization factor(IL-10,PPAR-γ)and decrease the expression of TLR7 gene in chicken alveolar macrophages.These results suggest that this may be one of the reasons why NDV Ex promotes NDV infection in vivo.In summary,this study revealed the important role of NDV Ex in the process of infection in vivo and its regulatory mechanism,providing a new idea and direction for further research on the mechanism of exosome action,and enriching a new theoretical basis for further interpretation of NDV infection and pathogenic molecular mechanism.