| Background:In the past decade,the world has witnessed the prevalence of emerging infectious diseases,such as Ebola,Zika,and the resurgence of Monkeypox virus,as well as the continued pandemic of the COVID-19,which posed a serious threat and far-reaching impact on global public health and social economy.Over the past 70 years,more than 80% of emerging infectious disease outbreaks have been caused by zoonotic or vector-borne pathogens,of which wild animals are important reservoirs for many zoonotic pathogens,and ticks are the vectors of more than 80 new tick-borne diseases.Particularly after the outbreak of the SARS-Co V-2,the research into pathogens carried by wild animals has become the focus of attention in order to trace the source of SARS-Co V-2.In some cases,this wildlife may increase the likelihood of spillover of zoonotic pathogens into humans,leading to epidemic spread.To effectively control the infectious disease epidemic,it is essential to study the epidemiology of pathogens carried by wild animals and their parasitic vectors.On the other hand,continuous surveillance for potential zoonotic pathogens,early detection and identification of unknown pathogens is also the key to effective prevention and control of emerging and re-emerging infectious diseases.The southeast coastal area of China is an important strategic location,developed economy,dense population,and frequent material flow.The natural environment in this area is suitable for the reproduction of a variety of medical organisms.It is an epidemic area and focus of multiple zoonotic diseases,seriously affecting the health of local military and civilian populations.Especially when the troops are marching,camping and performing exercises in this area,they are vulnerable to serious threats from zoonotic diseases.Strengthening surveillance,identification and potential epidemic risk assessment of pathogens carried by people,wild animals and their parasitic ticks in this region,and preventing cross-species transmission of wild animal-derived and tick-borne(especially hedgehog and tick)pathogens.On the other hand,understanding the basic information and transmission rules for important zoonotic pathogens in the region may improve the region’s response and control capabilities against novel and sudden infectious diseases,and prepare for defense against sudden biological threats in this strategic direction.In order to deeply understand the background information of biological threat factors in the southeast coastal area,studies on the genomics,epidemic distribution,genetic characteristics,and potential pathogenicity of key zoonotic pathogens carried by small wild animals and their parasitic ticks have been conducted in this area.Methods1.Sample collection and identification: Select representative pathogen monitoring outposts along the southeast coast as sampling points,and conduct continuous sampling in the region from 2017 to 2022.Use glue boards to capture rodents,and use nets to capture bats in artificial tunnels and caves.Small wild animals are collected through monitoring outposts near local forestry departments and mountains.Ticks are mainly collected on the surface of animals,The collected samples were then subjected to morphological and molecular biological population identification.2.Macrogenomics analysis: Extract sample RNA,purify and enrich it,construct a macrogenomics sequencing library,and sequence it using the MGISEQ-2000 instrument.Based on bioinformatics software,complete sequencing sequence classification,pathogen reads assembly and annotation.3.Pathogen detection: Extract sample nucleic acid,design pathogen detection primers,and use ordinary/nested PCR to detect important zoonotic pathogens in 1096 small wild animals and tick samples collected from 12 cities and counties of 6 provinces along the southeast coast.Based on ELISA technology,1786 human serum samples collected from 8sentinel hospitals in the southeast coast were tested for five important zoonotic specific Ig Gs.4.Genetic characteristics analysis of pathogens: Based on bioinformatics software such as MAGE 7.0,RDP4,Simplot,and Tree Map2.0b,we analyzed the structure of the pathogen genome,genetic evolution,gene recombination and transmission laws.5.Pathogenicity analysis of pathogens: Five potential zoonotic pathogens from hedgehogs infected suckling mouse,rats and different cells,and their pathogenic effects were observed.The morphology and infection of pathogens were observed by ultrathin edge electron microscopy and fluorescence in situ hybridization,and the pathological changes were observed by histological sections.Results:1.Metagenomic studies on pathogens carried by small wild animals and their parasitic ticks.1.1 Macrogenetic sequencing of viruses carried by small wild animals and ticks.High-quality reads range from 270 to 980 million reads were obtained from the macro-gene sequencing results of six wild animals and their parasitic ticks.After overlay splicing,the range of sequences available for comparison was 80～200 million reads,of which 0.05～4million reads(0.07%～2.37%)were viruses.A total of 37 viral families had been annotated,including 21 mammalian viral families,of which hedgehog was the most viral family among all wild animals in this study.Hedgehog had annotated to 15 mammalian viridae,including Hantaviridae,Arenaviridae,Coronaviridae,Phenuiviridae,Adenoviridae,Picornaviridae,Herpesviridae,Circoviridae,Polycipiviridae,Poxviridae,Retroviridae,Anelloviridae,Papillomaviridae,Mimiviridae and Paramyxoviridae.Compared with their parasitic host hedgehogs,ticks have been annotated with 9 mammalian viridae,and 7 viral families have been found in hedgehogs.Only Flaviviridae and Reoviridae are independently carried in ticks.1.2 High-throughput sequencing of pathogens of new tick-borne diseases.A large number of reads were obtained in 6 small wild animals and ticks,such as Rickettsiaceae,Anaplasmaceae,Coxiellaceae,and Bartonellaceae,among which Rickettsiaceae,Anaplasmaceae,and Coxiellaceae accounted for the largest proportion of ticks.2.Molecular and seroepidemiological investigation of important zoonotic pathogens.2.1 Pathogen screening.A total of 10 pathogens(coronavirus,arenavirus,SFTSV,hantavirus,dicipivirus,Rickettsia,Ehrlichia,Anaplasma,Coxiella,Bartonella)were detected from 1096 samples of wild animals and ticks.It was found that hedgehogs and their parasitic ticks carried the most types of pathogens.Hedgehogs carried the above 9 pathogens except Bartonella,and ticks carried Rickettsia,Anaplasma,Ehrlichia and Coxiella burnetii were detected.Arenavirus and Coxiella burnetii were detected in mice,and in turtledoves.Wild chickens were found to carry arenavirus and Coxiella burnetii,while wild ducks and rabbits were found to carry arenavirus and Coxiella burnetii,respectively.This study is first time to find that hedgehogs carry seoul virus,arenavirus,and Rickettsia,as well as turtledoves,pheasants and ducks carry arenavirus.This result expands the types of viruses and their hosts carried by wild animals and parasitic ticks.The ELISA results showed that the total positive rates of specific Ig G antibodies against Coxiella burnetii,Rickettsia,Anaplasma,Scrub typhus,and SFTS were 2.91%,4.30%,4.0%,3.35% and 2.40%,respectively.The positive rates of specific Ig G antibodies against Coxiella burnetii,Rickettsia and Scrub typhus in the sera of people in Fuzhou and Zhangzhou regions were obviously higher than those in other regions(P<0.05).2.2 Discovery of new pathogens.A new type of Candidatus Rickettsia xuyiensis XY-2and Coxiella burnetti(XYHT29)have been found in hedgehogs and ticks,a new type of Anaplasma phagocytophilum variant(xyn32ph-1)has been found in Rattus flavipectus and Haemaphilus longicornis,and a new type of Candidatus Cryptoplasma sp.(xyn113cr-1)has been found in Haemaphilus longicornis.2.3 Coinfection phenomenon.Multiple pathogenic infection were found in both ticks and their host hedgehogs,with the presence of four pathogenic composite infection in a single tick,such as Rickettsia,Anaplasma,Ehrlichia and Coxiella burnetti,as well as three tick-borne pathogens and one zoonotic virus co-infection in a single hedgehog,which may be the result of mutual infection between the tick and the host during blood-sucking.3.Analysis of genetic characteristics of important zoonotic viruses3.1 Genome-wide analysis.Hedgehog-derived coronavirus,STFV,hantavirus and dicipivirus belong to Merbecovirus coronavirus,STFV subtype C2 of Phlebovirus genus,S2 subtype of seoul virus and hedgehog dicipivirus of Dicipivirus genus,respectively.The arenavirus carried by hedgehogs belongs to a new type of arenavirus,and the arenavirus carried by rattus norvegicus belongs to a new mutant strain of Wenzhou arenavirus.3.2 Analysis of Genetic recombination.Based on the analysis of RDP4 and Simplot software,it was found that hedgehog-derived coronavirus,hantavirus,dicipivirus and arenavirus showed extensive gene recombination,except for STFV.3.3 Analysis of Cross-specific transmission.Based on Tree Map2.0 software to compare the evolutionary relationship between viruses and host genes,it was found that hedgehog-derived coronavirus,hantavirus,STFV and arenavirus have the characteristics of different degrees of cross-host transmission.4.Pathogenicity analysis of important zoonotic pathogens4.1 Cell and suckling mouse infection experiments.Using Vero-E6,BHK-21,MDCK and HEK 293 T cells to isolate hedgehog-derived coronavirus,seoul virus,STFV and arenavirus failed.At the same time,after 14 days of infection with the four hedgehog-derived viruses,the tissue nucleic acid test also showed negative results.However,the new type of Coxiella burnetii was successfully infected with suckling mouse,and positive results of the new type of Coxiella burnetii were detected in the spleen tissue of the infected suckling mouse.The results of in situ hybridization of the spleen of positive mice showed that the new type of Coxiella burnetii was mainly visible in the cytoplasmic region of the cells,indicating that it successfully infected suckling mouse.4.2 Infection experiment in rat.The infection of SD rats with Coxiella burnetii was successful.The nucleic acid test in the liver and spleen tissues of the infected rats was positive.The electron microscope observation showed that the pathogen particles could be detected,and there were no detailed lesions in the liver and spleen tissues of the infected rats.Conclusion:1.The first study on the pathogen spectrum of small wild animals and their parasitic ticks in the southeast coastal region of China has obtained high-quality pathogen spectrum data,enriched the understanding of wild animals and parasitic ticks as pathogen banks,meanwhile found and identified multiple new zoonotic pathogens and mutant strains.2.Nine species of zoonotic pathogens have been detected in hedgehogs,and multiple pathogen co-infection phenomena have been found in hedgehogs and their parasitic ticks,indicating that hedgehogs are important hosts or carriers of multiple zoonotic pathogens and play an amplified role in the transmission of tick borne pathogens.3.Focused on the analysis of the genetic evolution and cross-species transmission of five important zoonotic pathogens,it was found that the five zoonotic viruses have extensive genetic recombination phenomenon and the risk of cross-host transmission in different degrees.4.The study found that there were different degrees of infection among people in Southeast China,such as Coxiella burnetii,Spotted fever group rickettsiae,Anaplasmosis,Scrub typhus and SFTS.It is necessary to continue to strengthen the monitoring and detection of important zoonotic pathogens in this area.5.The preliminary analysis shows that the risk of cross species transmission of hedgehog derived coronavirus,STFV,seoul virus,and arenavirus is relatively low,while the new type of Coxiella burnetii from hedgehog has a certain potential for cross species transmission.In summary,based on macrogenomic sequencing,nested PCR,ELISA screening and bioinformatics analysis,this study has researched the pathogens carried by small wild animals and their parasitic ticks in the southeast coast,discovered multiple new pathogens and mutant strains,and preliminarily established a background database of the levels of pathogens carried by ticks and small wild animals in the southeast coast,providing background information for responding to zoonotic infectious diseases in the region.In particular,the preliminary identification of the spectrum of pathogens carried by hedgehogs and their parasitic ticks,as well as the ecological status of hedgehogs in their transmission,provides a strong scientific basis for early warning and prevention and control of pathogens carried by hedgehogs and their parasitic ticks,and lays a good foundation for responding to sudden biological threats in the region. |
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