Analysis Of Bronchoalveolar Lavage Fluid Viral Community In Patients With Fever Of Unknown Origin

Fever of unknown origin(FUO)is a common clinical syndrome.There are many diseases can cause FUO,which can be generally classified into infectious diseases,non-infectious diseases,neoplastic diseases and other diseases.There are many literatures in the world on the statistical analysis of fever of unknown causes,the results showed that infectious diseases are the main cause,mainly bacterial infection,but virus infection also presents a rising trend.For virus detection,traditional immunological methods and molecular biological methods have played an important role,especially PCR technology,with good accuracy and sensitivity,has become the gold standard for many viruses nucleic acid detection,but can only detect the known sequence of the virus.Due to the wide distribution of viruses,only 0.1%of all viruses are known to be pathogenic,and there are still a large number of unknown viruses in nature.At the same time,viruses themselves have a high degree of variability,which makes it impossible for most traditional methods to detect pathogens quickly and accuratelyIn recent years,with the progress of the second-generation sequencing technology and the development of bioinformatics technology,viral metagenomics emerged and became an important means for human to study new viruses.Through the use of random hexamer primers,viral metagenomics can reverse transcription and amplification of all viral nucleic acid sequences in specific samples,so as to detect both known and unknown viruses in samples However,this method is very sensitive to the infection of background nucleic acid in samples and the environment.In order to explore the relationship between the viral community in bronchoalveolar lavage fluid(BALF)and fever of unknown origin,this study used the method of viral metagenomics to analyze the viral community in alveolar lavage fluid collected from people’s hospital affiliated to jiangsu universityThe main research contents and results of this project are as follows(I)Composition of viral community in alveolar lavage fluidAfter the analysis of 56 samples,it was found that the virus community in alveolar lavage fluid mainly included:Herpesviridae 0.9%,Anelloviridae 19.4%,Ascoviridae 0.3%,Asfarviridae 0.3%,Baculoviridae 0.6%,Caliciviridae 1.8%,Circoviridae 11.1%,Coronaviridae 1.2%,Geminiviridae 0.3%,HERV 10.3%,Iridoviridae 1.8%,Marseilleviridae 0.3%,Mimiviridae2.4%,None 25.0%,Parvoviridae 2.1%,Phycodnaviridae 11.2%,Picornaviridae 0.6%,Poxviridae 5.0%,Retroviridae 3.2%,Sphaerolipoviridae 0.3%,and Virgaviridae 2.4%Based on the sequence number related to each virus,there were 2207 sequences related to Anelloviridae,accounting for 45.3%of the total sequence number,Circoviridae for 1.3%,Phycodnaviridae for 1.9%,HERV for 1.4%,Poxviridae for 28.1%,Virgaviridae for 13.5%,None for 6.9%(336)and Picornaviridae for 0.1%(5)Analysis on the viral community of alveolar lavage fluid indicated that:(1)Anelloviridae,Poxviridae,Circoviridae,Phycodnaviridae,None,etc.constituted the community of alveolar lavage fluid virus;(2)Anelloviridae are distributed in four libraries,and virus species account for 19.4%of the total virus community,and the number of sequences related to them is significantly higher than that of other viruses.(3)Circoviridae and the Phycodnaviridae have a large proportion of viruses,but the number of related sequences is small,on the contrary,the relatively small proportion of Poxviridae has a large number of sequences;(4)The number of sequences related to Virgaviridae is large,but it only exists in library 5;(5)There are many common respiratory viruses in the library(Ⅱ)Research of Torque teno virusFour strains of Torque teno virus,one of the most common viruses in the human body,were isolated from samples of alveolar lavage,and its infection may be associated with some diseases,but the pathogenic mechanism is unclear.In this study,four sequences of TTV were obtained,among which the longest sequence was 2918 bp,mainly including ORF1(389-2593 bp),ORF2(51-515 bp),ORF3(2070-2540 bp)and ORF4(2401-2784 bp).Based on this sequence,nested PCR primers were designed to confirm 80 samples and a total of 9 positive samples were found,with a positive rate of 11.3%,while 2 positive samples were found in 40 negative controls.Phylogenetic trees were constructed based on the ORF1 amino acid sequence of the virus,and analysis showed that the four TTV strains clustered with Torque teno virus from human plasma,with identity between 89 and 94%(Ⅲ)Research of rhinovirusRhinoviruses are one of the common viruses that cause respiratory tract infections.Although there is a small proportion of rhinoviruses in the library,they were also studied to understand their association with unexplained fever.A total of 8 positive samples were confirmed by nested PCR,with a positive rate of 10.0%.Phylogenetic tree analysis based on the relatively conserved nucleic acid sequences in the VP4/VP2 region showed that 4 strains belonged to human rhinovirus type A and 3 strains belonged to human rhinovirus type B,respectively clustered with the rhinoviruses from human nasopharyngeal swabs,with the identity above 95%Conclusion:(1)the virus community in BALF samples was polymorphic,in which Anelloviridae were dominant;(2)Torque teno virus is related to fever of unknown.Combining with the research results,it can be inferred that inflammatory environment caused by fever and low immune function can promote the proliferation of TTV(3)Because of the low proportion and titer of rhinoviruse,the association with fever is not well explained.