Epidemiologic Study On Hemorrhagic Fever With Renal Syndrome In Shandong Province

1. BackgroundHemorrhagic fever with renal syndrome (HFRS), known as epidemic hemorrhagic fever (EHF) in China, is caused by Hantavirus (HV). It is a natural focus infection disease which results in high fever, hemorrhage, renal function damage in clinic and affects people seriously. China is the country most seriously affected by HFRS and the number of the cases accounts for 90% of the total reported cases in the world. The case fatality rate was as high as 30% at the epidemic period. The HFRS case of Shandong Province was first reported in Yutai County, Jining in 1962. The incidence rates were increasing gradually since 1975 and reached to the highest in 1986. 41443 HFRS cases were reported and accounted for 50% of the total reported HFRS cases in China in that year. The incidence rate was 53.69 per 100,000. Since then, the number of the reported HFRS cases in Shandong Province was the highest, which accounted for 1/3 of total cases in China. The situation is far serious.There seemed to be an epidemic cycle about 10 years in Shandong by analyzing the tendency of HFRS. The epidemic peaks arose in 1986 and 1995 separately. The next epidemic peak maybe arises in the coming years according to the regularity. Whereas some fluctuations of HFRS epidemic in Shandong had occurred, the epidemic situation became unstable.It is high time to strengthen the surveillance analysis of HFRS and HV study in Shandong to detect the HV genovariation and find the possible new type HV strains as early as possible. It is necessary to prepare for the prevention of the coming epidemic situation. At the same time rapid detection and genotype method study of HV should be strengthen to adopt perfect clinical treatment and perform large-scale epidemic survey.2. Objectives2.1 To study the time, place and population distributions of the HFRS and their corresponding trends from 1994 to 2006 in Shandong Province.2.2 To study the category, ecology feature, beasts' density and HV carrying conditions of the HFRS hosts in Shandong and their corresponding tendency.2.3 To study the nucleotide sequence characterization and phylogenetic analysis of hantaviruses isolated in Shandong Province and their subtype distribution and homology comparison.2.4 To explore the rapid detection and genotype method of HV.3. Materials and Methods3.1 Patients The corrected epidemiological data of HFRS were obtained through routine internet reporting system of Shandong Province. The data were analyzed with Excel and SPSS11.5 software. Furthermore line and bar charts were drawn according to data. Geographic information systems (GIS) was also used to draw the distribute map of the HFRS cases in Shandong Province.3.2 Hosts The data of category composition, ecology feature, beasts' density and virus carrying conditions of the HFRS host animals came from the surveillance results of the HFRS surveillance spots in Shandong from 1994 to 2006. The data were analyzed by Excel and SPSS11.5 software.3.3 Molecular epidemiological studies of HV3.3.1 HV antigen detection of rat lungs and part of the HFRS patients serum Indirect immunofluorescence test (IFA) was applied to detect the HV antigens of the rat lungs and part of the HFRS patients sera.3.3.2 HV IgM antibody detection of some HFRS patient's sera MacELISA was applied to detect the HV IgM antibody of part of the HFRS patient's sera.3.3.3 RNA extraction Total RNAs of HFRS patient's sera and rodent lungs were extracted separately by single-step method of RNA extraction with acid guanidinium thiocyanate-phenol-chloroform.3.3.4 RT Nested PCR Partial M segments of HV RNAs were amplified with reverse transcription nested polymerase chain reactions (nested PCR) by using hantavirus genotype specific primers which were part of the G2 encoding region (2003nt～2302nt) of M segments.3.3.5 Sequence Products of nested PCR were purified using a Gel Extraction Kit and sequenced on a Model 377 DNA Sequence System with the same primers used for PCR amplification.3.3.6 The nucleotide homologies and phylogenetic analysis Alignment and comparative nucleotide sequence analysis were carried out and phylogenetic analysis was performed using DNASTAR. The sequences used in the phylogenetic analysis included sequences of HV isolates in the present study, sequences of previously epidemic HV isolates in Shandong and other representative HV sequences from GenBank. Statistical analysis was performed using student's t test. Values were compared between the homologies of HV from patients and those from rodents in Shandong Province.3.3.7 PCR/SSCP (single strand conformation polymorphism,SSCP) According to the S gene fragment of strain HV76-118 and SR-11, a pair of oligonucleotide primers (both a couple of inner primers and outer primers) that were complementary to the S gene fragment were designed. The total RNAs were extracted from HFRS patients sera and the partial S segments of HV were amplified from the RNAs with RT nested PCR by using inner primers and outer primers. The nested PCR products were analyzed by SSCP.4. Results4.1 Patients There were conspicuous peaks of the incidence and death of HFRS in spring and autumn in Shandong Province. The autumn peak heightened in recent years and came ahead of schedule. The fatality rates had increased since 2003 and reached 1.11% in 2005. The HFRS cases mainly appeared in Luzhongnan mountainous areas. The incidence rate had inceased in Ludongnan hilly areas and decreased in Luxibei plain areas as the whole incident rate in Shandong had decreased in recent years. The number of HFRS cases in male was more than in female cases from 1995 to 2006, which 67.21% cases were male and 32.79% cases were female, so the ratio was 2.05 : 1. The number of HFRS dead cases in male was also more than in female during the same period, which 71.82% cases were male and 28.18% were female, so the ratio was 2.55 : 1. The number of incidence cases in male hand increased appreciably and the female dead cases hand increased too. The cases' age was focus on 20-59 years old. More and more people older than 50 years reported to be infected with HV in recent years. Peasants were the main population of all kinds of cases, which accounted for 81.55% incidence cases and 82.84% dead cases. 4.2 Hosts The dominant rats in residential areas were mainly Rattus norvegicus (59.58%) and Mus musculus (32.39%). Apodemus agrarius (32.39%) and Rattus norvegicus (26.65%) were the dominant rats in field areas. The composition of rats in residential areas and field areas were different significantly (x~2=3356.588, P=0.000). In recent years the proportions of the Mus musculus increased and Rattus norvegicus decreased in residential areas, while the proportions of Apodemus agrarius decreased and Rattus norvegicus increesed in field areas. The diversity of ecological community was highest in Luzhongnan mountainous areas (0.6870) and the uniformity of the ecological community was highest in Ludongnan hilly areas (0.7038). Both the diversity and uniformity of the ecological community were lowest in Jiaolai plain (0.4337&0.4949). 9848 animals were detected from 1994 to 2006 in which 318 were HV antigen positive including 225 Rattus norvegicus (70.75%), 42 Mus musculus (13.21%), 29 Apodemus agrarius (9.12%), 10 Cricetulus triton (3.14%), 6 Sorex araneus (1.89%) and 6 Cricetulus barabensis (1.89%). The dominant infected rats in residential areas were Rattus norvegicus and Mus musculus, and the dominant infected rats in field areas were Apodemus agrarius. The beasts' density and virus carrying index had changed with the different seasons and reached the peak in spring and autumn.4.3 Molecular epidemiological studies of HV4.3.1 IFA Lung tissues from 572 rodents captured during 2004-2006 at the HFRS monitoring sites were examined by IFA, in which 20 were HV antigen positive including 2 from Apodemus agrarius and 18 from Rattus norvegicus.4.3.2 MacELISA 35 positive results were obtained by using MacELISA among 48 sera samples from patients who were clinically diagnosed as HFRS. The positive rate was 72.92%.4.3.3 RT Nested PCR Partial M segments of HV RNA extracted from 23 acute HFRS patients sera and 20 HV antigens positive rodent lungs were amplified with RT nested PCR using HV genotype specific primers. Three samples including 2 were from HFRS patients and 1 was from Apodemus agrarius were only nested PCR-positive by HTN primers (HPK HP2). Thus they should be classed as Hantaan virus (HTNV). However, 28 samples including 13 were from HFRS patients and 15 were from Rattus norvegicus were only nested PCR-positive by SEO primers(SP1,SP2) which should be classed as Seoul virus (SEOV).4.3.4 Sequence results 22 sequences of the partial M segment of the G2 region (2003nt-2302nt) were obtained by analyzing the sequence electropherogram data. One from a patient was HTNV. Ten from patients and eleven from Rattus norvegicuses were SEOV.4.3.5 Sequence analysis results HV nucleotide homologies in Shandong from 1984 to 2006 showed 67.1%～100%. The nucleotide homologies among HTNV isolates in Shandong were 78.1%～98.7%, while the homologies among SEOV isolates in Shandong were 93.7%～100%. There were at least 3 subtypes HTN (H2,H5,H9) and 2 subtypes SEO (S2,S3) in Shandong Province. There were differences significantly between the homologies of the SEOV from patients (96.89%±1.64%) and those from rodents (97.92%±1.11%) in Shandong Province by the student's t test (t=-3.02, P=0.003).4.3.6 PCR/SSCP 41 positive results were obtained by using RT Nested PCR from 48 sera samples from patients who were clinically diagnosed as HFRS. 33 were SEO type and 8 were HTN type according to the SSCP results.5. Conclusions5.1 The incidence rates of HFRS in Shandong had decreased gradually from 1994 to 2006 and the ratio of cases in autumn increased. The case fatality rate had raised since 2003. The center of high incidence rates of HFRS had switched from Luxibei to Ludongnan in Shandong. More and more people older than 50 years were reported to be infected with HV and died of HFRS in recent years. The female dead cases of HFRS increased.5.2 The ratio of Mus musculus in residential areas had increased from 1994 to 2006 in Shandong Province. The diversity of ecological community was highest in Luzhongnan mountainous areas and the uniformity of the ecological community was highest in Ludongnan hilly areas. The beasts' density and virus carrying index had changed with the different seasons and reached the peak in spring and autumn.5.3 HTN and SEO coexisted in Shandong with SEO being the prevalent genotype. There were at least 3 subtypes HTN (H2,H5,H9) and 2 subtypes SEO (S2,S3) in Shandong Province. H5 and S2 were the first reported subtypes in Shandong. HTNV had no predominant subtypes while S3 was the predominant SEO subtype in Shandong Province.5.4 The genetic variability among HTNV was higher than that among SEOV and SEOV were also in constantly genetic variability resulting in new subtype arising despite of their relatively stable heritage. The homologies of SEOV from rodents were higher than those from patients. HV distributions showed geographical clustering within individual HV types in Shandong. The distributions of HV subtypes in Luzhongnan mountain area were most complicated.5.5 RT-Nested PCR was more sensitive and specific than MacELISA in HFRS patients' sera detection which could provide reliability evidences in viral infection, especially to those infected within 1 week and the positive rate could be 100%. SSCP could type HV conveniently. It was practicable to early diagnosis and treatment decision making in clinic and epidemiologic practice.