| The mucosa of vertebrates is a particularly dynamic environment in which trillions of commensal microorganisms exist,known as the microbiota,which plays an important role in many biological functions including growth enhancement,nutrition,development,and metabolism.The microbiota has been found to be closely related to the host’s immune system,as it induces the occurrence of immune responses and enhances immune susceptibility.In response,the host mucosal immune system has evolved multiple means to maintain its symbiotic relationship with the microbiota.Teleost fish as one of the early vertebrates,have evolved both innate and adaptive immunity due to evolutionary pressures and can therefore protect themselves against pathogens residing in the aquatic environment and maintain mucosal microbiota homeostasis.Notably,unlike the mammalian nasopharynx,the nose and pharynx of teleost are two separate compartments that are not related due to the lack of a choana,forming nasal-associated lymphoid tissue and pharyngeal mucosa-associated lymphoid tissue.Studies have shown that the nasal and pharyngeal mucosa tissues of teleost have an immune response against pathogens,and the symbiotic bacteria in the nasal and pharyngeal mucosa tissues have also been identified in rainbow trout.However,the relationship between virus-mediated mucosal immune response and microbiota remains unclear.In this study,we successfully constructed an infection model by intraperitoneally injecting common carp(Cyprinus carpio)with spring viremia of carp virus(SVCV).Transcriptome sequencing and 16 S r RNA gene sequencing were performed on the nose and pharynx tissues of common carp at different time points after infection.The specific experimental contents and results are shown as follows:1.A less lethal concentration of the SVCV was administered to common carp via injection,then the common carp began to manifest significant clinical symptoms 3 days after infection,including exophthalmos,pale gills,loss of scales,and caudal fin bleeding.Moreover,the virus was also detected in the nose and pharynx of infected common carp,which could invade EPC cells and make them appear obvious CPE.All of which indicated that the SVCV can successfully invaded the nasal and pharyngeal mucosa tissues after intraperitoneal injection.The number of SVCV copies was detected in the nose,pharynx,spleen,and blood in the control and infected groups at 1,4,7,14,and 28 dpi by q RT-PCR.It was found that the viral load increased first and then decreased over time.2.In control groups and infected groups at different time points,the nasal and pharyngeal tissues of common carp were sectioned with paraffin wax and stained,and the different parts were measured and counted.We found that the width of the lamina propria in the nasal villi gradually enlarged with the increase of infection time and decreased at 28 dpi,while the width of the pharyngeal epithelium was showed to be significantly thinner than the control fish and recovered at 28 dpi.Moreover,the number of mucus cells in the olfactory epithelium and pharyngeal epithelium were increased first and then decreased.Using quantitative real-time PCR(q RT-PCR),we measured the relative expression levels of antiviral genes,antimicrobial peptide genes,immunoglobulin genes,and inflammatory genes in the nose and pharynx of common carp.Finally,we identified obvious histopathological changes and related gene expression changes following viral infection.3.The nose and pharynx samples of the control group and the SVCV infection group of 4 and 28 dpi were sent for transcriptome sequencing.The results showed that the expression level of antiviral genes,inflammatory genes,and antigen presentation genes were most significantly upregulated at 4 dpi.Interestingly,there was also a certain degree of high expression of antibacterial related genes,such as serum amyloid A protein(SAA)and galectin.Moreover,associated with bacterial infection in the three Pattern Recognition Receptors(PRRs)pathways were also activated.Therefore,we concluded that SVCV infection induced secondary bacterial infection on nasal and pharyngeal mucosal surfaces of carp.4.Same samples as in RNA-Seq were sent for 16 S r RNA gene sequencing,we found significant differences in the composition of microbiota on the surface of nasal and pharyngeal tissues at the phylum,order,and species levels compared with the control group at 4 dpi,meanwhile,there was a significant increase in opportunistic pathogens,such as Pseudomonadales and Acinetobacter.We believe that this might be due to the disruption of microbial homeostasis on the mucosal surface and mucosal tissue damage caused by virus invasion,which allowed opportunistic pathogens to invade the tissue,resulting in secondary bacterial infection. |
