| ObjectivesTo clarify the situation of natural infection of hantavirus on small mammal in part of commensal rodent plague foci in Yunnan Province and the influences of factors such as region,habitat,murine species and sex on the infection of hantavirus on small mammal,to explore the type and evolutionary variation characteristics of hantavirus on small mammal in part of commensal rodent plague foci in Yunnan Province,and to assess the risk of pathogenicity and prevalence for hantavirus infection in humans for providing scientific basis for effective prevention and control of the occurrence and prevalence of hantavirus disease.Methods1.Sample source: 488 lung samples of small mammals were collected from 16 natural villages in three regions of commensal rodent plague foci in Yunnan Province from July to August 2019,and were used as experimental objects.2.RNA extraction from lung specimens: 50 ~ 100 mg lung tissue was grinded and then used to extract total RNA through magnetic beads method.The extracted RNA was detected by ultramicro spectrophotometer,and the RNA with unqualified concentration and purity was re-extracted.RNA quality control: RNA concentration ≥ 50 μg/m L and RNA purity(A260/A280)between 1.9 and 2.1 were considered to be up to standard.3.PCR amplification and nucleic acid sequencing: The one-step dual real-time fluorescence quantitative PCR was used for the positive screening of two types of hantavirus of HTNV and SEOV for all specimens.The positive control of hantavirus was donated by Yunnan Institute of Endemic Diseases Control & Prevention.The S-segment of hantavirus of positive specimens was amplified and sequenced by ordinary PCR.4.Homology and phylogenetic analysis: The biological analysis software such as National Center for Biotechnology Information NCBI(BLAST)website and DNASTAR(Meg Align)were used for nucleotide and amino acid sequence homology of the successfully sequenced sequences.The MEGA-X software was applied to construct the phylogenetic tree and perform genetic evolution analysis.5.Data collation and analysis: R software was applied to statistically analyze the data.Chi-square Test and Fisher’s Exact Test were used to compare the infection status of hantavirus in different murine species,genders,regions,and habitats.The influencing factors were analyzed,and the testing level was α = 0.05.ResultsThe 488 small mammals captured belong to 3 orders,5 families,8 genera,and 16 species,respectively.There are two dominant murine species,of which Rattus tanezumi accounts for 50.00%(244/488)and Suncus murinus accounts for 30.74%(150/488).The constituent ratio of each of the other non-dominant murine species does not exceed2.87%.The infection rate of hantavirus on small mammal in part of commensal rodent plague foci in Yunnan Province is 2.46%(12/488).The infection rate of hantavirus on small mammal of 3.45%(8/232)for Lianghe County is the highest,followed by 1.58%(3/190)for Mangshi City and 1.52%(1/66)for Mile City.There is no statistical difference in the infection rates of hantavirus among small mammal in the three regions(c2 = 1.471,(49)= 0.558).A total of 3 types of small mammals were infected with hantavirus,among which Rattus tanezumi,as the dominant species,accounts for an absolute proportion.Hantavirus was also detected in Rattus nitidus that does not belong to the dominant species.In the positive specimens,Rattus tanezumi accounts for 66.67%(8/12),which is the important host of hantavirus,and Suncus murinus and Rattus nitidus both account for 16.67%(2/12).There is no statistical difference in the infection rates of hantavirus between the dominant murine species of Rattus tanezumi and Suncus murinus(c2 =0.004,(49)= 0.949).There is also no statistical difference in the infection rates of hantavirus between dominant and non-dominant murine species(c 2 = 0.000,(49)=1.000).4.The infection rates of hantavirus on female and male small mammals are 3.05%(8/262)and 1.77%(4/226),respectively.There is no statistical difference in the infection rates of hantavirus between female and male small mammals(c2 = 0.833,(49)=0.361).5.Different habitats were divided into four categories.The infection rates of hantavirus on small mammal for different habitats are 5.26%(5/95)in bushes,2.22%(4/180)in the environment near residents,1.57%(2/127)in grass and 1.16%(1/86)in mountain forests.There is no statistical difference in the infection rates of hantavirus among small mammals in different habitats(c2 = 3.389,(49)= 0.331).There is also no statistical difference in the infection rates of hantavirus in small mammals between the two habitats((49)> 0.05).6.Genetic evolution and phylogenetic analysis show that the hantavirus gene sequences obtained in this study cluster in the subgroup to which the SEOV genotype belongs and have a relatively distant evolutionary relationship with the representative reference strains such as DOBV,HTNV,SNV,PUUV and ANDV.The obtained YNLH-K40 and YNLH-K53 are aggregated to form an independent branch,which is the closest to the SEOV S5 subtype(GOUV)branch composed of strains such as GOU3,Longquan Rn09-132 and ZJ5 originating from Longquan City,Zhejiang Province,China,but the highest nucleotide homology between these sequences is only 88.5%.Meanwhile,NCBI(BLAST)analysis indicates that the highest homology of YNLH-K40 and YNLH-K53 with the published sequences is less than 90%.According to the recommendations of the International Committee on virological nomenclature on the typing of hantavirus,it can be considered that YNLH-K40 and YNLH-K53 belong to the new gene subtype of SEOV.The two sequences YNLH-K40 and YNLH-K53 found this time are closely associated with the previously described GOUV variants,but they are in a separate evolutionary sub-branch.This phylogenetic pattern reveals the new genetic variation in the newly sequenced strains,and the epidemic strains have regional characteristics.Conclusions1.The infection rate of hantavirus on small mammal in part of commensal rodent plague foci in Yunnan Province is 2.46%(12/488),including 1.52%(1/66)in Mile City,1.58%(3/190)in Mangshi City and 3.45%(8/232)in Lianghe County.The small mammal infected with hantavirus include Rattus tanezumi(dominant species),Suncus murinus(dominant species)and Rattus nitidus(non-dominant species).The discovery and monitoring of hantavirus host animals in Yunnan Province should be further strengthened.2.The infection rate of hantavirus in small mammal near residents is 2.22%.It is necessary to be vigilant and take correspongding measures to reduce the risk of contact between residents and small mammal.The infection rate of hantavirus in small mammal in natural vegetation environments such as grass,shrubs and mountain forests range from 1.16% to 5.26%.It is necessary to provide prevention and education to outdoor work and tourism groups.3.A new SEOV subtype,named YNLH-K40 and YNLH-K53,has been discovered,which is highly likely to cause human infections and diseases and even spread within certain regions.In addition to further promoting the improvement of the hantavirus gene database in Yunnan Province,attention should also be paid to the potential pathogenicity of newly discovered hantavirus type. |
PDF Full Text Request