| As animal viruses infect humans and cause serious public health and safety incidents,viral interspecies transmission,especially the host shift,where a pathogen crosses the species barriers and jumps from its original host into a novel species,causes increasing attention.Viral covert mortality disease(VCMD),caused by covert mortality nodavirus(CMNV),is an emerging disease,which caused severe economic losses of shrimp aquaculture in China recently.In order to investigate the natural hosts of CMNV in aquaculture animials and in the pond culture system,the samples of Litopenaeus vannamei and Mugilogobius abei from the shrimp farming ponds attacked by VCMD,Carassius auratus from the drainage channel of a shrimp farm attacked by VCMD,Acanthopagrus schlegeli from outlet water of shrimp farming ponds,and Paralichthys olivaceus cultured in the same farm with shrimp attacked by VCMD were collected for CMNV tests by reverse transcription loop-mediated isothermal amplification(RT-LAMP),reverse transcription nested PCR(RT-nPCR),in situ hybridization(ISH)and transelectron transmission microscope assays.To further study and confirm the infectivity and pathogenicity of CMNV in fish,CMNV artificial infection of the zebrafish as model organism,molecular biological assay,histopathological analysis,ISH assay and transmission electron microscopy(TEM)assay were conducted to reveal the main target tissues,the histopathological changes of CMNV infection in fish.1.CMNV nucleic acid detection,ISH and TEM assays were conducted to analyze the infection of CMNV in L.vannamei collected from a farm in Weifang,Shandong Province which was doubted as suffering VCMD.The result of both RT-nPCR and RT-LAMP indicate that the samples of L.vannamei were CMNV positive.Histopathological analysis showed that the nuclei of the epithelial cells of hepatopancreas tubules of the diseased shrimp were significantly pyknosis,and virus inclusion bodies were seen in the cytoplasm of the epithelial cells,and the muscle tissue coagulated and necrosed,and blood cells infiltrated between the muscle cells.Positive signals were verified in tissues of hepatopancreas tubules and skeletal muscle by ISHassasy with CMNV probes.Under TEM,CMNV particles were further visualized in the cytoplasm of the epithelial cells of hepatopancreas tubules in the CMNV positive samples diagnosed by ISH.2.The natural infection of CMNV in M.abei collected from a shrimp farming pond with occurrence of VCMD was investigated.The result of RT-nPCR indicated that the M.abei sample was CMNV positive.Histopathological analysis showed that CMNV infection in M.abei could induce structural damage in the ganglion cell layers in the eyes,nervous tissue vacuolation and karyopyknosis in cerebellum of brain,extensive skeletal muscle necrosis and hemocytes infiltration.Positive signals were verified in eye,brain and skeletal muscle by in situ hybridization(ISH)with CMNV probes.The findings suggested that CMNV,a typical Alphanodavirus originated from shrimp,could infect M.abei naturally and cause pathological damage of the target tissues.3.The natural infection of CMNV in C.auratus collected from the drainage channel of a shrimp farm attacking by VCMD was investigated.The results of CMNV RT-nPCR showed that the C.auratus sample could be amplifed and produced the expectant target gene fragment of CMNV RdRp gene.Histopathological analysis showed that CMNV infection in C.auratus could cause nervous tissue vacuolation in brain and obvious karyopyknosis occurred in the granular cells of corpora bigemina and pyramidal cells of cortex.ISH analysis showed CMNV positive signals could be observed in pathological damage region of the brain and cardiac muscle.TEM analysis verified the nervous tissue vacuolation in brain and the presence of CMNV particles in the cardiac muscle.The results indicated that CMNV could cross the species barriers to infect fish and caused pathological damage in the target tissues.4.The systematic analysis of natural infection of CMNV in the A.schlegeli collocted from outlet water of shrimp farming ponds in Zhejiang Province was conducted.The results of RT-nPCR indicated that the A.schlegeli sample could be amplifed and produced the expectant target gene fragment of CMNV RdRp gene.Histopathological analysis result indicated that the CMNV-positive fish showed vacuolation of nervous tissue in the eye and brain,structural damage in the ganglion cell layers in the eyes,mild lytic necrosis of cardiac muscle,muscle fibers arrangement loosening and lymphocyte infiltration,the hepatic sinusoids expanding and severe congestive symptoms,hepatocyte degeneration and cytoplasm vacuolization with karyopyknosis.The results of ISH analysis showed positive reactions occured in tissuesof the eye,brain,heart,and liver of the diseased fish.The result of TEM analysis demonstrated the presence of CMNV particles in cardiac muscle and granule cells.All the mentioned results indicated that the diseased A.schlegeli was infected naturally by CMNV and the infection induced the nerve injury in the tissue of eye and brain,pathologic changes in liver,heart and muscles.5.The natural infection of CMNV in P.olivaceus cultured in the same farm with shrimp attacked by VCMD in Jiaonan Shandong Province was investigated.Through the RT-nPCR test targeting the RdRp gene,we found that 29% of the fish sampled were CMNV positive.The RT-PCR amplicons were sequenced and aligned to the CMNV RdRp gene of shrimp and were found to have a 98% identity.The histopathological examination indicated that the CMNV-positive fish showed vacuolation of nervous tissue in the eye and brain and,also,extensive necrosis of cardiac muscle.ISH analysis showed positive reactions in tissues of the eye,brain,heart,liver,spleen,and kidney of the fish.TEM analysis showed the presence of CMNV particles in all of the above mentioned tissues except for brain.The results demonstrated that the samples P.olivaceus were infected naturally by CMNV,which caused severe pathological lesion in the main tissue of disease individauls.6.Taking the model organism,zebrafish,as the experimental object,the main target tissues of CMNV and histopathological changes caused by CMNV infection,as well as the pathogenicity and lethality of CMNV to zebrafish was systematically analyzed.After intraperitoneal injection of CMNV into the zebrafish,infected individuals showed abnormal swimming behaviors including unbalanced swimming movement,vertically or horizontally spiraling.Some diseased individuals showed symptom of prominent abnormalities of the eye.The cumulative mortality rate of the infected group was 48% post-injection 7 days.The histopathological result indicated that vacuolation of nervous tissue occured in the eye and brain of the diseased individuals,coagulation necrosis and hemocytes infiltration happened in the muscle tissue.ISH analysis showed positive reactions in tissues of the retina of eye,brain,muscles of the fish.TEM examination indicated the presence of CMNV particles in eye and brain.All of the above mentioned tissues except for brain of the diseased zebrafish.The results demonstrated that CMNV could infect freshwater zebrafish artificially in laboratory and caused the severe tissues damages and high motality of the infected individuals.The results of natural infection of CMNV in M.abei,C.auratus,A.schlegeli and P.olivaceus demonstrated that CMNV,an alphanodavirus originally isolated from shrimp,possesses capability of crossing the species barriers and infecting vertebrate including both fmarine fish and brackish water fish.The results from the CMNV intraperitoneal injection of zebrafish indicated that CMNV could infect zebrafish and caused relative high cumulative mortality rate of the infected individuals,which reminded us to pay close attention to the high risk of CMNV to use fish as intermediate or new host as well as potentially spread or cause epidemic among cultured marine fish and brackish water fish. |
