Genotype And Pathogen Characteristics Of Hantavirus In Main Endemic Areas Of HFRS In Hebei Province

Objective: To investigate the gene characteristics and variation of hantavirus(HV)which lead to hemorrhagic fever with renal syndrome(HFRS).To reveal the genotypes of HV carrying by rodent in hemorrhagic fever epidemic areas of Hebei province,and to provide clues for the prevention and treatment of HV related diseases.Methods:1 According to the prevalence and distribution of HFRS in Hebei Province,we selected a coutry-level city of Shijiazhuang,a coutry-level city of Baoding,a coutry of Cangzhou,a coutry of Handan,a coutry of Tangshan and a coutry of Chengde as the focus areas.2 Capture rats in the residential area and the field.The density and species composition of HV host animals were summarized.3 Rat lungs were homogenized and total RNA was extracted.4 The rat lung specimens were detected by 4 channel detection of 8 kinds of HV using TaqMan probe multiple fluorescence quantitative RT-PCR.Hantaan virus(HTNV),Seoul virus(SEOV),Puumala virus(PUUV),Dobrava virus(DOBV)were the first group,and Tula virus(TULV),Black creek canal virus(BCCV),Andes virus(ANDV),Sin Nombre virus(SNV)were the second groups.5 Genotypes of HV in rat lung specimens were detected by consensus degenerate hybrid oligonucleotide primers(CODEHOP)RT-PCR.6 HV positive rat lung specimens were chosen for further amplified by PCR using SEOV specific primers designed for the Gc gene fragment,and the amplified products were sequenced.7 The sequencing results were analyzed by homology and phylogenetic tree.Results:1 In 2015 and 2016 two years of 6 areas,the field average rat density was 0.44%,and residential average rat density was 2.77%.The density of residential area was higher than that of field area(?2=317.995,P<0.001).2015 to 2016 in southern Hebei(a county-level city of Shijiazhuang,a county of Handan)the average rat density was 5.36%.The average mouse density was 2.58% in central Hebei(a county of Baoding,a county of Cangzhou),the density of the rats in eastern Hebei(a county of Tangshan)was 1.92%,and the density of the rats in northern Hebei area(a county of Chengde)was 6.18%.According to the chi-square test,the density of rodents in northern Hebei area and southern Hebei was higher than that in eastern Hebei and central Hebei areas(?2 = 336.29,P <0.001).The residential rat density in different seasons was slightly higher than autumn or basic with autumn.2 Between 2015 and 2016,in 6 areas,a total of 1228 rats were identified,belonging to 4 genera and 4 species,there were Rattus norvegicus(1043),Mus musculus(179),Tscherskia triton(5),Apodemus agrarius(1).Rattus norvegicus was the dominant in residential areas,accounted for 84.12% of the number of the total catch in the residential area;Rattus norvegicus was the major species in field,accounted for 92.50%;Overall,Rattus norvegicus were dominant species in 6 areas of Hebei Province.3 TaqMan probe multiplex fluorescence quantitative RT-PCR was used to detect 1228 rat lungs,and 35 HV positive samples were detected.4 1228 samples of rat lung were PCR-amplified using the CODEHOP system,and 29 rat lung specimens appeared characteristic bands.5 The positive rate was about 2.85%,and the virus was SEOV.A total of 1108 samples were detecte in the residential area,33 were positive,and the positive rate of the mice was about 2.98%.A total of 120 mice were detected in field,and 2 were positive,the rate of mice with virus was 1.67%,The positive rate of mice in residential area was higher than that in field(?2=37.78,P<0.05).Among 35 HV positive specimens,there were 29 Rattus norvegicus,5 Mus musculus,with the poison rate of 2.78%,2.79%,respectively,and 1 Apodemus agrarius(the specimen size is too small to calculate the positive rate).Mus musculus and wild type Apodemus agrarius carrying SEOV,proved that the phenomenon of "host overflow" appeared in this virus.In different regions,the poisoning rat in southern Hebei was 2.13%,the poisoning rat was 0.34% in central Hebei,17.81% in eastern Hebei and 5.88% in northern Hebei.The results of chi-square test showed that(?2 = 2513.13,P <0.001): eastern Hebei was higher than other areas.6 The SEOV type specific primers for Gc gene fragments were amplified by PCR in 35 lung samples of HV positive specimens,and 8 rat lung specimens appeared characteristic bands.7 The seven L fragments were obtained for nucleotide amplification and homology comparison,the nucleotide homology was 94.0%~98.3%.Their nucleotide constitutions were similar with SEO type of HV strains(ZT10 from Zhejiang and 89-30 from Korea).The phylogenetic tree of L fragment showed that the 7 rat lung specimens were all in the branches of type HV SEO,and the genetic relationship was close.It can be concluded that the rat lung specimens of all the 7 samples belonged to type SEOV.8 Typical specimens were chosen to sequence,the result of comparing of nucleotide homology of Gc.8 rat lung specimens nucleotide homology was 98.1%~99.7%,which were in the same clade with BjHD01 strain in Beijing,HBQ20 strain and HBQ43 strain in Hebei province,GM04-38 strain and ZB8 strain in Shandong province,and they had close genetic relationship of each other,all belonged to the S3 subtype.Conclusions:1 The residential density of rat and the positive rate of HV in hemorrhagic fever epidemic area of Hebei province was higher than that in field.Rattus norvegicus was the dominant host animals in HFRS main endimic areas in Hebei Province.2 SEO was the dominant genotype which was carried by host animals in epidemic areas in Hebei,no other type of HV was detected in rat specimens.3 The host animals in epidemic areas in Hebei carried single subtype,only S3 subtype.4 Mus and the wild type Apodemus agrarius carryed SEO type HV,there is a “host overflowing”.