| Transmissible viral proventriculitis(TVP)mainly causes symptoms such as weight loss,fecal material,and proventricular enlargement in chickens.The disease was first discovered in1978 by Dutch scientist Kouwenhoven and spread to countries around the world.Since the first case in China was reported in Jiangsu Province in 1994,it was then exploded in various provinces and cities across the country.The disease can significantly increase the feed to meat ratio,reduce the production of chicken,and bring huge economic losses to the broiler breeding industry.TVP has become the main problem in broiler breeding.The etiology of TVP has not been clear,although many domestic and foreign researchers have isolated a variety of viruses,such as reticuloendotheliosis virus,infectious bronchitis virus,infectious bursal disease virus,reovirus,avian leukosis virus subgroup J,Marek's disease virus,chicken infectious anemia virus,chicken proventricular necrosis virus,etc.It is impossible to use the isolated virus to reproduce the disease.Based on previous studies and the results of a large number of epidemiological investigations,it is speculated that the disease is caused by an unknown new virus.In order to identify the unknown new virus,typical TVP cases that exclude the 8 TVP-associated viruses mentioned above.Then,TVP model was reproduced.PacBio three-generation sequencing platform was used for high-throughput sequencing of viral metagenomics,isolating and identifying new virus,and then study the pathogenesis of the virus.In order to obtain TVP cases that exclude known viruses associated TVP,336 samples of TVP from 80 broiler farms were tested by histopathology and PCR.A case of TVP that excluded the 8 TVP-associated viruses mentioned above were selected for further study.To reproduce TVP by the selected case,the proventriculus homogenate was injected into1-day old SPF chicks.The TVP was successfully reproduced.The infected chickens showed typical symptoms of TVP.The infected chickens were tested by PCR for known TVP-associated viruses.The results showed that all 8 known TVP-associated viruses were negative.In order to identify the virus,the infected proventriculus was observed under transmission electron microscopy.The result showed that virions were circular or icosahedralsymmetrical,without capsules,and the diameter in size was 26-28 nm.Viral metagenomic high-throughput sequencing was then performed using the PacBio three-generation sequencing platform and BLAST alignment with the 2017 NCBI latest virus library.The alignment sequences were identified as a same virus,the Gyrovirus 3(GyV3).The full-length sequence of the virus was amplified using inverse PCR,and the full-length sequence of the virus was finally obtained to be 2356 bp,which was named GyV3-SDAU-1 strain.The GyV3-SDAU-1 strain contains three overlapping open reading frames encoding three proteins,capsid protein VP1,backbone protein VP2,apoptotic protein VP3.NCBI looked for a total of30 viral genome-wide sequences of the Circoviridae and the Anelloviridae to construct a phylogenetic tree.The results showed that GyV3-SDAU-1 has the closest homology with GyV3-FecGy and belongs to the Gyrovirus genus of Anelloviridae.The GyV3 is a single-stranded circular DNA virus.It was first isolated and identified in children's feces of acute gastroenteritis in Chile and Hong Kong in 2012,indicating that the virus is closely related to gastroenteritis.According to Koch's rule,it was proved that GyV3 can cause TVP.Firstly,a retrospective epidemiological investigation was conducted on 336 cases of infectious gastroenteritis and 102 normal chicken cases.The results showed that the GyV3 positive rate was 12.5%(42/336)in 336 cases of TVP,0%(0/102)in normal chickens.It indicates that the GyV3 is ubiquitous in flocks with TVP,but does not exist in healthy chickens.In order to culture the GyV3,the epithelium of proventriculus of chicken embryo were successfully prepared in vitro.and the epithelium were infected by GyV3 infected chicken proventriculus homogenate.The cells were collected for PCR.PCR detection showed that GyV3 was positive,and 8 known proventriculitis-associated viruses were negative,indicating that the virus can replicate in cultured epithelium cells.Then,the cultured GyV3 was inoculated into SPF chickens,and the infected chickens showed weight loss,proventricular enlargement,proventricular nipple swelling,lymphocytes infiltration in proventricular mucosa and lamina propria,showing typical TVP symptoms,and the GyV3 can be isolated again from the diseased chicken,in accordance with Koch's law,which proves that the GyV3 can indeed cause TVP.In order to study the pathogenicity of GyV3,the cultured GyV3 was inoculated into SPF chickens.The lesions were observed by gross necropsy and histopathology.The tissues were collected from 7 days old.Significant inflammation occurred,peaking after 14-21 days,and symptoms of 35 days of age were alleviated,which coincided with the age of onset of TVP infield cases.In addition to the typical inflammation,the infected chicken can also cause anemia and immunosuppression in chickens.The effects of GyV3 on the pathogenicity of SPF chickens by intraperitoneal,intravenous,subcutaneous and oral injection were studied.It was found that different inoculation methods showed the same pathological changes and course changes.This indicates that the GyV3 can be infected in a variety of ways.The effects of GyV3 on the pathogenicity of SPF chickens at different ages(7 days,14 days,21 days,28 days old)were studied.It was found that chickens of different ages showed the same pathological changes and course changes.The GyV3 can infect chickens at different ages.In order to explore the viral loads of GyV3,eight timepoints of infected chickens were tested by absolute fluorescent quantitative PCR(1,2,3,5,7,14,21,28 days after infection).The virus content in the organ of 29 chickens.The results showed that the organs with the highest viral loads were adrenal and bone marrow,followed by nerves,ovaries and pith.Adrenal and neuroviral levels indicate that the cause of proventriculitis is that the virus invades the adrenal glands and nerves,leading to inflammation of the proventriculit is by fluid regulation and neuromodulation.After the GyV3 infection,the hematopoietic stem cells and immature immune cells in bone marrow were poorly developed,causing anemia and immunosuppressive symptoms.At the same time,the high viral loads of the ovary suggest the possibility of GyV3 vertical transmission.The high viral loads of the feathers suggest the possibility of horizontal transmission through the feathers.In order to explore the pathogenisis of GyV3,the interaction between VP1 of GyV3 and proteins of host cell was studied by immunoprecipitation combined with mass spectrometry,and four signal molecules of signal transducers and activators of transcription 3,T cell receptor,caspase recruitment domain family member 9 and casein kinase 1 involved in the invasion,host immunity,the signaling recognition,activation of innate immunity and specific immunity pathway.In summary,this study used the high-throughput sequencing of viral metagenomics to isolate and identify the new virus-GyV3 from TVP case that excluded known proventriculitis-associated viruses,and reproduced TVP indicating that GyV3 is direct casue of TVP.In addition to proventriculitis,GyV3 also causes anemia and immunosuppression.The viral loads of GyV3 test showed that the GyV3 highly replicated in the adrenal gland and bone marrow.Four molecules of signal transducers and activators of transcription 3,T cell receptor,caspase recruitment domain family member 9 and casein kinase 1 of host cells interact with capsid protein VP1 of GyV3.Finally,this study provides a base knowledge for GyV3 pathogenesis and control. |
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