Genovariation Study Of Hantavirus In Main Endemic Areas Of HFRS In Hebei Province

Objective: To understand the epidemic characteristics of hemorrhangicfever with renal syndrome (HFRS) main endemic areas of Hebei Provinceover the years, explore the genotype and genovariation of host animals carriedhantavirus(HV), in order to guide the prevention and control for this disease.Methods:1The epidemiological data of HFRS in cities of Hebei Province during1996and2013were collected. The epidemiological data include thecharacteristics of category composition, density, the virus carried rate of rat;2Capture rats and take rat lungs in sterile environment. Rat lungs weresliced with freezing microtome. Then HV positive lungs were screened withIFA after they were fixed by cold acetone;3Total RNA were extracted from positive lungs and strains. Real timePCR was applied for genotyping of HV;4The entire M segments and partial M segments were amplified byRT-PCR, PCR amplification products were purified. The amplified productswere cloned into pMDl9-T vector. Plasmids were purified and sequenced;5The molecular evolution method and software were used to calculatethe nucleotide and amino acid homology and phylogenetic trees wereconstructed.6Statistical software of SPSS13.0was applicated to process and analyzethe statistical.Results:1Tangshan and Qinhuangdao cities were located in the northeast ofHebei Province, whose incidence were both significantly higher than averageincidence in Hebei Province since2001. Both cities are now main endemicareas of HFRS in Hebei Province. 2The average density of rat was2.26%in Tangshan and Qinhuangdaocities from2004to2013, of which residential area was2.75%and field areawas1.16%.They showed a increased trend over time. The virus carried rate ofrat was10.29%in average, of which residential area was11.23%and fieldarea was2.96%.There were obvious difference in residential area and fieldabout density and virus carried rate of rat.Through classifying and identifying the composition of rodent inTangshan and Qinhuangdao cities from2009to2013, Rattus norvegicus wasthe dominant host animals not only in residential area but also in field. Therewas significant difference between residential area and field’s composition ofrodent.3HV positive lungs and strains of Hebei were conducted with real timePCR. The result indicated that HV,21positive specimens, all belonged to SEOtype and HTN type have not found.4The typical specimens were chosen to sequence for G1and G2segments. The nucleotide homology of G1segment (217-573nt) was95.5%-100%, among which93HBQ3and93HBQ4were95.5%,95.8%withHBQ17/2012respectively. TS-2/2004and HBQ20/2007, HBT49/2013andHBT50/2013were100%. Their nucleotide constitutions were similar withR22strain (94.5%-96.3%), But when compared with76-118, the nucleotidehomology was low (69.9%-71.8%). The nucleotide homology of G2segment(2002-2301nt) was high (97.4%-100%), among which93HBQ3and93HBQ4were both97.4%with HBQ5/2012. TS-2/2004, HBQ20/2007andHBQ43/2009, HBQ7/2013and HBQ17/2012were all100%. When comparedwith R22and76-118strains the rates of nucleotide homology were90.8%-92.7%and71.3%-72.6%respectively. It showed that the variation ofHV was small.5The phylogenetic tree of G1(217-573nt) and G2(2002-2301nt)segments were constructed by DNASTAR package. The phylogenetic tree ofG1segment displayed that QHD-1/2004, TS-2/2004, HBT-3/2012,HBT14/2012, HBT49/2013, HBT50/2013, HBQ20/2007, HBQ43/2009, HBQ5/2012, HBQ17/2012, HBQ7/2013and BjHD01had closer geneticrelationship.93HBQ3,93HBQ4and ZT71, changchun had closer geneticrelationship. And all specimens and the reference strains were in the sameclade, which belonged to the S3subtype. The phylogenetic tree of G2segmentdisplayed that all14specimens and the reference strains were in the samebigger clade, which belonged to the S3subtype.93HBQ3,93HBQ4and ZT71,ZT10had closer genetic relationship. TS-2/2004, HBT67/2011, HBT69/2011,HBT-3/2012, HBT14/2012, HBT49/2013, HBT50/2013, HBQ20/2007,HBQ43/2009, HBQ5/2012, HBQ17/2012, HBQ7/2013and cp27c had closergenetic relationship. Although all specimens belonged to S3subtype，93HBQ3,93HBQ4had changed significantly.6The recombinant plasmid of M segment was constructed constructedand sequenced, the result showed that M segment sequence of all specimenswere3651nt in length. M segment consisted of3sections:5’non-codingregion (NCR), one open-reading frame (ORF) and3’NCR. The nucleotidelength of5’NCR and3’NCR was47nt and203nt respectively. The M segmentcontained a single ORF (nt48to3448) which encodes the protein precursor of1133amino acids (aa). As a member of HV, all specimens had distinctiveterminal complementary nucleotides that promote the folding of the viralgenomic segments into “panhandle” hairpin structures.711typical specimens were chosen to sequence, the result of comparingof nucleotide homology displayed that they have higher homology, bothtransition and transversion were found. The nucleotide homology of all11specimens was95.8%-99.8%, among which93HBQ3and93HBQ4was95.8%-97.0%with other9specimens. Both93HBQ3and93HBQ4was98.7%,and other9specimens themselves were higher (98.6%-99.8%). Moreover,their nucleotide constitutions were similar with SEO type of HV strains (R22,L99, BjHD01, Z37), the nucleotide homology was83.6%-99.2%. But whencompared with HTN type of HVstrains (Q32, Lee,76-118), the nucleotidehomology was low(70.0%-71.7%) respectively. It further showed that thevariation of HV was small. 8The phylogenetic tree of M segment was constructed by DNAStarpackage. The phylogenetic tree of M segment displayed that HBT-3/2012,HBT14/2012, HBT49/2013, HBT50/2013, HBQ20/2007, HBQ43/2009,HBQ5/2012, HBQ17/2012, HBQ7/2013and BjHD01had closer geneticrelationship.93HBQ3,93HBQ4and Z37, ZT10had closer geneticrelationship. But all11specimens and the reference strains were in the sameclade, which belonged to the S3subtype. The phylogenetic tree analysisresults of partial M segments were consistent with that of the whole Msegments.Conclusions:1Tangshan and Qinhuangdao cities are main endemic areas of HFRS inHebei Province, the endemic situation of which had a increased trend in recentyears. Rattus norvegicus was the dominant host animals in HFRS mainendimic areas in Hebei Province.2On the basis of genotyping and phylogenetic trees analysis, Hebeiprovince is a typical endemic area of SEOV. The subtype of S3was exit andS3was the major subtype.3The variation of HV in main endemic areas of HFRS in Hebei Provincewas small. The genome sequence was relatively conservative for homeotypeof M segment, but the same subtype in different virus strains exist obviousvariation.4The variation of HV had small within a short time, but a significantchange within a long time.