Investigation And Analysis Of Pathogens Carried By Host Animals In Heilongjiang And Yunnan Province,China

With the deterioration of the natural ecological environment and frequent personnel exchanges,emerging and re-emering infectious diseases continue to threaten the health of humans and animals.The timely detection and identification of pathogens is the key to effective control of infectious diseases.Wild animals are important natural hosts for a variety of pathogens.Under certain circumstances,pathogens can be transmitted to humans and other animals through direct or indirect contact,causing the spread of the epidemic.Heilongjiang and Yunnan provinces in China are rich in natural resources.A large number of wild animals live in the same ecological environment,which provides favorable conditions for the spread of pathogens among different animal species.The purpose of this study is to investigate and analyze the types and distribution of pathogens in small wild host animals in Heilongjiang and Yunnan provinces,to discover and isolate potential pathogens,and to grasp their genetic evolution laws,so as to provide a reference for rapid response to possible future related public health emergencies.Raohe County,Heilongjiang Province,Ruili City and Jinghong City,Yunnan Province were selected as sampling sites,and local small wild animals were collected by traps.The heart,liver,kidney,lung,brain and other tissues of animals were dissected.After the tissue samples were grouped according to animal type,sex,age and number,the Tri Zol method was used to extract nucleic acid and construct a library,then performed viral metagenomic sequencing.After removing the host information,bioinformatics analyses was used to perform species annotation,abundance,heat maps,principal component analysis maps,species cumulative curve,random forest maps,etc.,to compare pathogen composition among different species of animals in different regions,clarifying the spectrum of pathogens carried by local wild animals.For animal samples infected with Hantavirus and Beilong virus,tissue grinding fluids were inoculated to Vero cells and SPF chicken embryo for virus isolation.After specific primers were designed and segment sequences were obtained by PCR,the phylogenetic trees were constructed and clarified the evolutionary characteristics of Hantavirus and Beilong virus.The results of the study as follows:1.15 species of small wild animals such as Rattus norvegicus,Rattus flavipectus,and Suncus murinus were collected,and 570 tissue samples were obtained,which were divided into21 pools for virus metagenomic sequencing.After the host genomes were removed,1,363,138 sequences were obtained.The numbers of sequences annotated as viruses,bacteria,and fungi were 184,495(13.53%),1,164,824(85.45%),and 13,818(1.01%),respectively.The annotated viruses covered 33 families and 55 genera.The top 10 virus families in the virus spectrum accounted for more than 99.84%,mainly were Hantaviridae(120,667 entries,65.40%),Paramyxoviridae(50,751 entries,27.50%)),Hepatitis E virus family(10,275,5.56%),Picornavirus family(1,157,0.62%).Nineteen virus species were found in the samples of Rattus norvegicus in Heilongjiang Province,which were significantly higher than other animals,of which Hantavirus accounted for 70.82%.Hepatitis E virus(46.93%,43.48% and 6.04%)was found in Rattus flavipectus(YNRF1),Rattus flavipectus(YNRF2)and Rattus flavipectus(YNRF3),and Beilong virus(28.86%)was found in Rattus norvegicus RHRN1 samples.2.A total of 199 bacterial orders and 411 bacterial families were annotated for all animal samples.The top 10 orders for bacterial reads accounted for more than 88.87% of all objects,mainly Pseudomonadales(29.83%),Burkholderiales(16.08%),Enterobacterales(9.11%),Sphingomonadales(9.04%),Rhizobiales(6.38%),Propionibacteriales(6.11%),Xanthomonadales(3.80%),Lactobacillus(3.70%),Corynebacteriales(2.41%)and Clostridiales(2.37%).Among them,Rattus norvegicus carries the most types of bacteria,reaching 169 orders.Pseudomonas are mainly concentrated in Rattus norvegicus and yellow ferret,and Burkholderia are concentrated in Rattus norvegicus and squirrels.In addition,a large number of opportunistic pathogens have been found in Rattus norvegicus,such as Acinetobacter johnsonii,Delftia acidovorans,Moraxella osloensis,Shiga flexneri Shigella flexneri,Pseudomonas,Escherichia coli and so on.3.Three strains of Hantavirus were isolated from the Rattus norvegicus collected in Heilongjiang,belonging to the SEOV type and highly homologous to the sequence of the Shandong strain in 2017.Six Beilong virus strains were isolated from the Rattus norvegicus collected in Heilongjiang.They have high sequence homology with the Wuhan strain in 2019,but they are independent branches and may be a new type of Beilong virus.There were a large number of pathogens that may cause disease to humans and animals in small wild animals in Heilongjiang and Yunnan.For example,Rattus norvegicus in Heilongjiang carried Hantavirus and Beilong virus,and Rattus norvegicus and squirrel carried Pseudomonas.There are significant differences in pathogen spectrum among different animals in different regions,which may be caused by the combined influence of natural environmental factors and human activities.The isolated Hantavirus and the new Beilong virus strain have enriched the virus library carried by wild animals in China,updated the understanding of the types and distribution of viruses carried by wild animals in Heilongjiang and Yunnan,and provided a basis for subsequent research on the pathogenicity of related pathogens.The relevant conclusions of this study suggest that long-term dynamic monitoring of pathogens carried by wild animals is of great significance for the prevention and control of emerging infectious diseases.