Genomic Analysis Of Enteroviruses And Screening Of Their Potential Pathogenic Viruses In Chickens

There are many pathogenic microorganisms that can cause chicken disease,among which virus is the most important one.At present,the common clinical toxic diseases of chicken include avian influenza,infectious laryngotracheitis,infectious bronchitis,Newcastle disease,chicken pox,infectious bursal disease,infectious encephalomyelitis,Marek's disease and leukaemia,which have caused great losses to the chicken industry.However,in the daily chicken production,there are often unexplained disease infections.It is difficult to determine the pathogen,diagnose and prevent the disease quickly and timely by using conventional methods.Based on the second-generation sequencing,the virus macro genomic technology has been widely used in the detection of unknown pathogens.At present,the technology has been successfully used in the screening of new environmental viruses,the analysis of enterovirus groups of mammals and birds,and the exploration of the relationship between human diseases and pathogens.However,there are few reports about the macrogenome of chicken enterovirus.In this study,feces samples from two chicken farms in Anhui Province were collected,and a virus macro genomic library was established to analyze the composition of enterovirus community and screen for new viruses.The purpose of this study is to identify the dominant virus groups and their potential epidemic infectivity in different chicken farms,and to screen new viruses that may cause chicken diseases Epidemic virology,diagnosis and prevention provide theoretical support.The research contents are as follows:?1?fecal samples were collected from two chicken farms in Anhui Province?chicken leukaemia non purified chicken farm No.A and purified chicken farm No.B?,30 samples were collected from each farm,totally 60 samples were collected.Four libraries?Ast01,Ast02,Bst03 and Bst04?were constructed by using the library construction kit,and the library was purified and tested;?2?the original sequence of the library was processed and divided into Analyze the virus sequence number and virus group difference of different libraries;?3?carry out homology comparison and genetic evolution analysis on the potentially pathogenic virus sequence of the library;?4?identify and analyze the dominant virus screened from the library;?5?find out the potential recombinant virus through preliminary sequence comparison,use rdp4.0 to carry out gene recombination analysis and different recombinant sections the phylogenetic tree was constructed respectively;?6?based on the whole gene sequence of the recombinant virus,nested PCR primers were designed to investigate the epidemiology of 30 samples in field A.The results showed:?1?agarose gel electrophoresis showed that 4 libraries were qualified.?2?There were 46607 and 45497 virus sequences in Ast01 and Ast02,respectively.Coronaviridae accounted for the highest proportion,49.82%and 30.90%respectively,followed by unclassified viruses 25.20%and 28.95%,Picornaviridae 20.79%and 20.65%respectively.The number of virus sequences of Bst03 and Bst04 were 15951 and 17054,respectively.The proportion of unclassified family,Retroviridae and circoviridae was 40.75%and 57.87%,39.15%and 13.20%,9.07%and 10.78%,respectively.Among them,field A accounted for 41.01%of the coronaviridae,26.95%of the unclassified viridae and 20.72%of the micro RNA viridae;field B accounted for 49.31%of the unclassified viridae,26.09%of the Retroviridae and 9.93%of the circoviridae.?3?One strain of astrovirus,one strain of calicivirus,one strain of micro RNA virus and two strains of circovirus?smaco1 and smaco2?were found in field A.The similarity between astv and km254166 strain was 87.2%,and astv and mg846415 strain,mn732559 strain from Brazil were clustered into one strain.The similarity between calicivirus and jq347532 was 86.8%,and it was clustered with mn175552.The similarity between small double RNA virus and ku729767 strain from Hong Kong was63.4%.Smaco1 and smaco2 belong to different genera of circovirus.The cap structure of smaco1 is 59.6%similar to that of mh111124 strain from Vietnam,and the rep structure is located in the branch of porprismacovirus,which is 64.8%similar to that of mh500336 strain from Vietnam,but it is one with that of mh500333 strain from Vietnam.The cap structure of smaco 2 is located in the branch of bovismacovirus,which is 41.8%similar to km598409strain from the United States;the rep structure is an independent branch,which is 62.7%similar to mh111124 strain from Vietnam.A chicken leukosis virus?ALV?and a micro RNA virus were found in field B.the similarity between ALV and jf932002 was 96.3%.The similarity between mi RNA virus and lc123275 strain from America was 36.4%,but it became a branch independently and may be a new virus.?4?A 27718bp genome of avian bronchitis virus was obtained from Ast01,named ahysx-1,which encodes 10 open reading frames.The non-s gene segment of the whole genome is 98%similar to that of kx252787strain?IBV?,58%similar to that of S gene segment,but 79%similar to that of turkey coronavirus eu022526 strain,indicating potential recombination of the virus.?5?The results of rdp4.0 analysis show that ahysx-1 has recombination phenomenon,and the probability of recombination is p-value<6.2×10^-15,S gene segment,KX252787 strain as the main parent and EU022526 strain as the secondary parent;98%similarity between ahysx-1 and five infectious bronchitis viruses based on phylogenetic tree constructed in non-s region;74.8%-75.9%similarity between ahysx-1 and three Turkey coronaviruses and one pearl chicken coronavirus based on phylogenetic tree constructed in S region,which verified the reorganization results.?6?All the 11 positive samples of IBV were from field A,the positive rate was 36.7%.The results showed that there were 92,104 virus sequences in non purified A field of chicken leukaemia,mainly in coronaviridae;33,005 virus sequences in purified B field of chicken leukaemia,mainly in unclassified viridae;there were differences in virus group composition and virus richness between field A and field B,the virus sequences in field a were 2.79 times of those in field B,and the purification of chicken leukaemia was beneficial to reduce the distribution of pathogens in chicken farm Reduce the virus load of chicken.A potential pathogenic virus?ahysx-1?screened in field A was a recombinant chicken infectious bronchitis virus?IBV?.There is IBV infection in a chicken farm,which has a high risk of IB.