Antigenic and genetic properties of Seoul virus strains--a causative agent of hemorrhagic fever with renal syndrome

Data on the circulation of a new genetic variant of Seol virus, which was designated as "Vladivostok" (the name of the place where it was isolated)--VDV, were obtained, by using the antigenic and genetic analysis of hantavirus strains isolated from patients with hemorrhagic fever with renal syndrome (HFRS) and from brown rats, and described in the present paper. A complete identity of nucleotide sequences of the M-segment fragment of isolates obtained from HFRS patients and of those obtained from rats was demonstrated, which confirms the key role of brown rat as a source of infection.     

家鼠型肾综合征出血热病毒单克隆抗体的制备与鉴定

以家鼠型HFRS病毒R22株免疫BALB/c小鼠,取免疫鼠脾细胞与小鼠骨髓瘤细胞系Sp2/0-Ag14融合,获得了23株能稳定分泌HFRS病毒特异性McAb的杂交瘤细胞系。以18株不同型别的HFRS病毒株进行分析,结果表明上述McAb中有些为HFRS病毒组特异性的,有些为家鼠型特异性的。有2株McAb对家鼠型HFRS病毒株具有较高的中和活性,对HFRS病毒R22株感染乳鼠也有明显的保护作用。     

HLA-associated hemorrhagic fever with renal syndrome disease progression in slovenian patients

Major histocompatibility complex (MHC) class I and class II genes regulate the balance between appropriate aggressive responses and invading pathogens while minimizing the destruction of host tissue. Several studies have shown that in hemorrhagic fever with renal syndrome (HFRS) patients, the disease outcome is determined by a complex interaction between the virus and immunopathologic and human genetic factors. In Slovenia, the severity of the disease caused by Puumala virus (PUUV) is significantly lower than that of HFRS due to Dobrava virus (DOBV). We have determined 23 different HLA-B and 12 different HLA-DRB1 types in Slovenian HFRS patients. Comparison of HLA frequencies between healthy individuals and HFRS patients showed no strong association with the susceptibility for hantaviral infection. Significant associations were recognized when the patient group was separated according to the virus responsible for the infection. DOBV-infected patients have a significantly higher frequency of HLA-B*35 than PUUV-infected patients. For HLA class II genes, the biggest difference between the PUUV- and DOBV-infected groups of patients was in HLA-DRB1*13, where this phenotype was more frequent in PUUV-infected patients, especially in the severe form of the disease. HLA-B*07 could play a protective role in PUUV-caused HFRS in the Slovenian population. Our study shows diverse associations of HLA molecules with DOBV- and PUUV-induced HFRS, and therefore, we presume that different hantaviruses are presented differently through the same HLA molecules and that this might lead to either a more severe or a milder form of the disease. In line with this idea, we have noticed that HLA-B*35 might be a genetic risk factor for DOBV infection in the Slovenian population.     

Molecular Diagnostics of Hemorrhagic Fever with Renal Syndrome during a Dobrava Virus Infection Outbreak in the European Part of Russia

A large outbreak of hemorrhagic fever with renal syndrome (HFRS) occurred in the winter of 2006-2007 in a region southeast of Moscow in Central European Russia. Of the 422 patients with HFRS investigated in this study, 58 patients were found to be infected by Puumala virus, whereas as many as 364 were infected by Dobrava-Belgrade virus (DOBV). Early serum samples from 10 DOBV-infected patients were used for nucleic acid amplification, which was successful for 5 patients. Molecular analyses demonstrated that the causative hantavirus belongs to the DOBV-Aa genetic lineage, which is carried by the striped field mouse (Apodemus agrarius) as the natural reservoir host. Neutralization assays with convalescent-phase sera from these patients confirmed infection by DOBV-Aa; related viruses, such as the Dobrava-Slovenia virus (DOBV-Af) and the Dobrava-Sochi virus (DOBV-Ap), were neutralized at lower efficiencies. The clinical courses of the 205 patients enrolled in the study were found to be mostly mild to moderate; however, an unexpectedly high fraction (27%) of patients exhibited severe illness. One patient died from kidney failure and showed symptoms of generalized subcutaneous hemorrhage. The results provide molecular, serodiagnostic, and clinical evidence that DOBV-Aa is a common pathogen in East Europe that causes large outbreaks of HFRS.Hemorrhagic fever with renal syndrome (HFRS) is an infectious disease caused by hantaviruses in Asia and Europe (16, 20) and most probably in Africa (13; our unpublished results). In the Americas, the autochthonous hantavirus species cause the hantavirus (cardio)pulmonary syndrome (24, 26). Hantaviruses carry trisegmented negative-strand RNA genomes and belong to the family Bunyaviridae. Different rodent species are known to be the natural hosts of the currently identified hantaviruses pathogenic for humans (16, 20). The viruses are transmitted to humans by aerosolized rodent excreta. Shrews were shown to be alternative hantavirus hosts (14, 31); however, the clinical significance of shrew-borne hantaviruses is still unclear.The demonstration of viral nucleotide sequences in specimens derived from patients with HFRS gives a clue to the involvement of a particular virus in the infection and allows its detailed molecular phylogenetic characterization. Because of the short-term viremia typical for human hantavirus infections, viral genetic material can best be amplified from blood or plasma during the first days after the onset of clinical symptoms (16). In contrast, serotyping of neutralizing antibodies is suggested to be most significant when convalescent-phase sera from the patients are used (19).HFRS cases in the European administrative regions of Russia are contributing to 97% of the total number of HFRS cases registered in Russia. Until recently, the majority of HFRS cases in European Russia were caused by Puumala virus (PUUV) and considerably fewer (mainly sporadic cases) were caused by Dobrava-Belgrade virus (DOBV) infections. However, during the period from 1991 to 2006, three large DOBV-associated HFRS outbreaks were registered in the central regions of European Russia (34). A detailed investigation of the 2001-2002 outbreak revealed that the striped field mouse (Apodemus agrarius) was the virus reservoir and that the A. agrarius-borne DOBV lineage (DOBV-Aa) was the causative infectious agent (15, 33).Three DOBV genetic lineages, DOBV-Aa (6, 11, 15, 33), DOBV-Af (1, 2, 3, 7, 9, 22, 23), and DOBV-Ap (15, 33), have so far been demonstrated by molecular methods to cause HFRS in humans. The Saaremaa virus can be considered a fourth DOBV genetic lineage (21); however, it has not been detected in HFRS patients. Moreover, it has been claimed to be a separate virus species by its discoverers (25).In the winter of 2006-2007, a large outbreak of HFRS involving 661 registered cases (according to official statistics of the Russian Ministry of Public Health) occurred in the Lipetsk region and the neighboring regions of Voronezh, Tambov, and Ryazan of Central European Russia. In the study described here, we investigated 422 of the cases to identify the causative pathogenic agent. By serodiagnostic methods, 364/422 patients were found to be infected by DOBV. For 205 of these patients, clinical data were collected and the specific clinical course of the disease was defined. Moreover, we amplified viral genetic material from the acute-phase sera of five patients and characterized the virus type as DOBV-Aa.     

中和试验发现西安地区为肾综合征出血热混合型疫区

目的 调查陕西西安地区汉坦病毒感染现状,分析当地肾综合征出血热(hemorrhagic fever with renal syndrome,HFRS)疫源地性质.方法 根据HFRS发病报告数据,分析西安HFRS的发病特点.通过荧光定量RT-PCR方法,检测西安HFRS疫区家鼠和野鼠携带汉坦病毒情况及病毒型别.微量中和试验确定西安本地HFRS病人、HFRS疫苗接种者和隐性感染者血清中的汉坦病毒中和抗体水平并分型,回顾性调查阳性感染者.结果 西安地区HFRS发病每年均有6-7月份的小高峰和10-12月份的大高峰;在当地642只家鼠和1 546只野鼠肺组织中检出汉滩病毒RNA136份;143份人血清中123份检测到汉坦病毒中和抗体,未检测到中和抗体的有20份.中和抗体阳性者中判定为汉滩病毒感染者92人,占74.80％;判定为汉城病毒感染者3人,占2.44％;不能区分病毒感染型别者28人,占22.76％,3例感染汉城病毒者分别为HFRS病人、疫苗接种者和隐性感染者,调查显示3例均为本地感染.结论 实验室和现场调查证实当地存在汉城型病毒本地感染,陕西西安地区是以汉滩病毒型为绝对优势的HFRS混合型疫区.     

Genetic Diversity of Apodemus agrarius-Borne Hantaan Virus in Korea

Hantaan (HTN) virus, the etiologic agent of clinically severe hemorrhagic fever with renal syndrome (HFRS), was first isolated in 1976 from lung tissues of striped field mice (Apodemus agrarius) captured in Songnae-ri, Kyungki-do, Korea. To clarify the genetic diversity and phylogenetic relationship among Korean strains of HTN virus, viral sequences of the partial S and M segments were amplified from lung tissues of 24 seropositive striped field mice captured between 1989 and 1998 at 11 sites in South Korea. The 771-nucleotide (nt) S segment sequences (coordinates 432 to 1202) of HTN virus strains from Yangju-kun differed by 10 to 40 nt (1.3 to 5.2%) from virus strains from Pocheon-kun, Songnae-ri and Nonsan-kun. Similar degrees of genetic variation were found in the G1 and G2 glycoprotein-encoding M segment. Phylogenetic trees, based on the partial S and M segments and generated by the maximum parsimony and neighbor-joining methods, demonstrated that virus strains from various geographic regions in South Korea showed a tendency to form two phylogenetic subgroups and were evolutionarily distinct from HTN virus strains from the Peoples Republic of China.     

Genetic analysis of hantaviruses and their rodent hosts in central-south China

Hantaan virus (HTNV) and Seoul virus (SEOV) are two major zoonotic pathogens of hemorrhagic fever with renal syndrome (HFRS) in Asia. Hubei province, which is located in the central-south China, had been one of the most severe epidemic areas of HFRS. To investigate phylogenetic relationships, genetic diversity and geographic distribution of HTNV and SEOV in their reservoir hosts, a total of 687 rodents were trapped in this area between 2000 and 2009. Sequences of partial S- and M-segments of hantaviruses and mitochondrial D-loop gene from 30 positive samples were determined. Our data indicated that SEOV and HTNV were co-circulating in Hubei. Phylogenetic analysis based on partial S- and M-segment sequences revealed two and three previously undefined lineages of SEOV, and a novel genetic lineage of HTNV, respectively. Four inter-lineage reassortment SEOVs carried by Rattus norvegicus and Apodemus agrarius were observed. It suggests that SEOV may cause spillover infections to A. agrarius naturally. The abundance of the phylogenetic lineages of SEOV suggested that central-south China was a radiation center for SEOVs.     

肾综合征出血热病毒50kD结构蛋白的抗原位点分析

采用23株抗HFRS病毒McAb,以放射免疫沉淀及ELISA等试验,证实HFRS病毒的50kD蛋白为该病毒的核蛋白(NP)。用抗NP的McAb对经亲和层析纯化的NP以ELISA阻断试验进行抗原位点分析,结果表明该蛋白与一般有囊膜病毒的NP相比,有两点明显的不同。一是HFRS病毒的NP上不仅有组特异性抗原位点,而且有型特异性抗原位点,两者都至少分布在2个区域,这些抗原位点及其分布区域之间有部分重叠或比较靠近。二是HFRS病毒的NP上可能存在中和抗原及血凝抗原位点。     

用碱性磷酸酶免疫斑点试验检测小鼠血清中的HFRS病毒抗体

将ＨＦＲＳ病毒陈株可溶性抗原吸附于硝酸纤维素膜上，建立了用碱性磷酸酶免疫斑点试验（ＡＰ－ＤＩＢＡ）检测小鼠血清中的ＨＦＲＳ病毒抗体的方法，并与应用辣根过氧化物酶的免疫斑点试验（ＨＲＰ－ＤＩＢＡ）作了比较。结果表明，ＡＰ－ＤＩＢＡ具有特异性；其敏感性高于ＨＲＰ－ＤＩＢＡ。该法可用于不同ＨＦＲＳ病毒株感染小鼠血清中抗ＨＦＲＳ病毒抗体的检测。     

肾综合征出血热病毒结构蛋白的纯化及免疫学特性分析

应用从肾综合征出血热（ＨＦＲＳ）患者血清中提取的ＩｇＧ与活化的Ｓｅｐｈａｒｏｓｅ４Ｂ偶联，制备亲和层析柱，用此亲和层析柱从ＨＦＲＳＶ感染的小白鼠乳鼠鼠脑中提取出２种分子量为６７０００和５５０００的病毒结构蛋白。经ＥＬＩＳＡ证明，此病毒结构蛋白能与ＨＦＲＳ患者血清ＩｇＧ及抗ＨＦＲＳ病毒的ＭｃＡｂ反应，效价为１６０。与６株抗ＨＦＲＳＶＭｃＡｈ试验表明，此病毒结构蛋白能与Ｈ７株反应。血凝试验证明，此病毒蛋白不能凝集鹅红细胞、鸽血球和人＂Ｏ＂型血红细胞。将纯化的病毒结构蛋白免疫家兔，证明其有较强的免疫原性，可刺激家兔产生特异性抗体；微量中和试验及乳鼠中和试验证明，中和效价为２５６，说明纯化的病毒结构蛋白具有中和抗原位点；免疫血清对感染乳鼠有一定保护作用。     

Puumala hantavirus in Slovenia: analyses of S and M segment sequences recovered from patients and rodents

In Slovenia, the co-existence of Dobrava and Puumala (PUUV) hantaviruses in a single endemic region has been demonstrated. This study presents selected Slovenian HFRS cases caused by PUUV combined with genetic analysis of viral genome sequences recovered from clinical specimens and tissue samples of Clethrionomys glareolus (bank voles). Serum samples from nine HFRS patients were included in the study. Rodents study sites were selected with regard to the HFRS cases. Partial S segment sequences were recovered from all nine patients and partial M segment sequences could be recovered from seven. Partial S and M segments sequences were also recovered from five C. glareouls captured at three different study sites. The sequences from Slovenian clinical specimens and rodent tissue samples belonged to the PUUV genotype and formed a distinct genetic lineage of PUUV. Human and rodent PUUV sequences located in the closest proximity to each other on the phylogenetic trees suggest genetic links between the human cases and the hantaviral strains circulating in natural foci of this zoonotic infection. Analysis of the complete S segment sequences recovered for two wild-type PUUV strains confirmed the existence of a distinct genetic lineage and also indicated a possible quasispecies type of Slovenian PUUV.     

Use of monoclonal antibodies for differentiating strains of the virus causing the hemorrhagic fever with renal syndrome

Using monoclonal antibody and animal immune sera the experiment confirmed the existence of antigenic relationships between the strains of virus of hemorrhagic fever with renal syndrome (HFRS) isolated in the USSR from Clethrionomys glareolus and members of all known serotypes of HFRS virus. Clear-cut differentiation was made from serotypes Apodemus and Rattus, and cross-relationships were shown between the strains isolated in the USSR and Prospect Hill virus (PHV), a member of the serotype Microtus isolated in the USA.     

Effectiveness of using cultured antigens for the serodiagnosis of hemorrhagic fever with renal syndrome by the immunofluorescence method

Clinical serological studies of hemorrhagic fever with renal syndrome (HFRS) demonstrated high effectiveness of indirect immunofluorescence procedure (IF) for serodiagnosis of this infection. The use of HFRS virus-infected VERO-E6 cells as the antigen for IF enhanced the sensitivity and specificity of the method as compared with the use of cryostatic sections of the lung tissue from wild rodents spontaneously infected with HFRS virus as the antigen. A technology for preparation of a polyvalent HFRS diagnosticum containing the antigens of two virus serotypes has been developed for serological diagnosis of HFRS in the European foci where the serological evidence of circulation of strains of serotypes 1 and 2 of HFRS virus pathogenic for man has been obtained. The specificity and sensitivity of detection of antibody to HFRS virus serotypes 1 and 2 by IF test using the polyvalent diagnosticum is equal to that in tests using individually tissue culture antigens of HFRS virus serotypes 1 and 2. It was found that in the patients' native sera antibodies of the IgM class are masked by antibodies of the IgG fraction, therefore the use of IF technique for detection of IgM antibody to HFRS virus aimed at early serological diagnosis of this infection is not effective.     

Serological analysis of hemorrhagic fever with renal syndrome (HFRS) patients in Far Eastern Russia and identification of the causative hantavirus genotype

Summary. Hemorrhagic fever with renal syndrome (HFRS) is endemic in East Asia and Europe. The disease is caused by several viruses belonging to the genus Hantavirus, including the Hantaan virus (HTNV), Seoul virus (SEOV), Dobrava Belgrade virus (DOBV), and Puumala virus (PUUV). Recently, HTNV-related viruses, Amur (AMR) and Far East (FE) genotypes were identified as causative agents of HFRS in Far Eastern Russia. To investigate the epidemiology of HFRS and virus transmission, we collected sera from 17 acute and 32 convalescent patients who were clinically diagnosed with HFRS in the Khabarovsk region of Far Eastern Russia, and detected anti-hantavirus antibodies using an ELISA that can differentiate the infected virus serotype using truncated hantavirus nucleocapsid protein antigen. Sixteen of the 17 acute phase patients had antibodies to hantavirus, and all the positive sera had higher optical densities for HTNV-specific antigen than for SEOV-, DOBV-, or PUUV-specific antigens. The partial M segment of the viral genome was amplified from blood clots from three acute patients by PCR. The nucleotide sequences had closer identities to the FE genotype (>96%) than to the prototype HTNV (88 to 89%) or AMR genotype (81 to 83%). A phylogenetic analysis found that the virus sequences from the patients clustered with the FE type, and were distinct from the AMR type. Thirty-one of 32 convalescent patient sera had antibodies to HTNV-specific antigen. These data suggest that our ELISA system can detect HTNV-specific antibodies to the FE type, which may be responsible for most of the HFRS in Khabarovsk.Received August 29, 2002; accepted March 10, 2003 Published online June 2, 2003     

A soluble and heat-resistant form of hantavirus nucleocapsid protein for the serodiagnosis of HFRS

Hemorrhagic fever with renal syndrome (HFRS) is a rodent-borne viral zoonosis characterized by fever, hemorrhagic manifestations, and renal disorder. The causative agent of HFRS has been identified as a hantavirus. Hantavirus nucleocapsid proteins have recently been shown to be immunodominant antigens in HFRS, inducing an early and long-lasting immune response, and their amino termini are sensitive tools for the detection of specific antibodies in HFRS patient sera. Previous work has demonstrated that the introduction of the acidic tail of alpha-synuclein (ATS) into heat-labile proteins protects them from heat-induced aggregation. In this study, the ATS peptide was introduced into the N-terminal antigenic portion of the nucleocapsid proteins (amino acid residues 1-70) of the Hantaan virus (HTNV-delta N) and Seoul virus (SEOV-delta N). The recombinant ATS-HTNV-delta N and ATS-SEOV-delta N fusion proteins were heat-resistant, and the proteins purified by heat treatment were immunoreactive to sera from patients with HFRS. Compared with sera from patients with leptospirosis and scrub typhus, sera from patients with HFRS showed much higher reactivity in ATS-HTNV-delta N- or ATS-SEOV-delta N-based IgG ELISAs. Immunoblotting analysis revealed that only sera from patients with HFRS specifically recognized the ATS-HTNV-delta N and ATS-SEOV-delta N, indicating that the ATS-HTNV-delta N and ATS-SEOV-delta N were highly purified species without any other immunoreactive proteins as contaminants. These data demonstrate that the ATS-HTNV-delta N and ATS-SEOV-delta N fusion proteins offer a safe and inexpensive source of pure and specific antigen for large-scale diagnosis and seroepidemiological studies of HFRS, and that ATS-fusion technology can also be utilized to solubilize other antigens that could be used for large-scale diagnosis and seroepidemiological studies of other diseases.     

Elevated levels of total and Puumala virus-specific immunoglobulin E in the Scandinavian type of hemorrhagic fever with renal syndrome.

In a previous study, it was reported that the total immunoglobulin E (IgE) level was increased in patients with hemorrhagic fever with renal syndrome (HFRS). The aim of the present study was to investigate whether specific IgE is synthesized during the course of the disease. For this purpose, an epsilon-capture enzyme-linked immunosorbent assay was developed. A total of 72 patients with HFRS caused by Puumala virus were studied. Three different control groups were included: 20 blood donors, 20 patients with other viral diseases (influenza A and B virus, acute Epstein-Barr virus, and acute cytomegalovirus infections), and 5 subjects with high levels of total IgE (median, 1,070 kU/liter; range, 773 to 5,740 kU/liter). The levels of total IgE were significantly higher during the acute phase of HFRS than those of blood donors (P < 0.01) and of patients with other viral diseases (P < 0.001). All patients developed a specific IgE response (median, 55 arbitrary units; range 24 to 123 arbitrary units) in the acute phase of the disease, whereas in the different control groups no specific IgE was detectable. Both total and specific IgE levels decreased during convalescence compared with those during the acute phase of HFRS (P < 0.001 and P < 0.001, respectively). In conclusion, we have shown that both total and specific IgE levels are increased in patients with HFRS compared with levels in patients with other viral diseases. The possible pathogenetic role of the specific IgE response in HFRS is discussed.     

微斑免疫酶技术在检测肾综合征出血热病毒及其特异抗体中的应用

本文报道一种简便且快速的方法—微斑免疫酶技术(MPIPA),检测Vero-E6细胞培养的肾综合征出血热(HFRS)病毒及其特异抗体,并与免疫荧光技术(IFA),酶联免疫吸附技术(ELISA)进行比较。结果,本法用于检测HFRS患者血清IgG抗体和抗HFRS病毒单克隆抗体(McAb),其敏感性介于ELISA和IFA之间;用于检测Vero—E6细胞培养的病毒,阳性结果比IFA和细胞病变(CPE)出现早。MPIPA具有简单,快速、敏感性高、特异性好,结果易判定诸优点,便于基层医疗单位推广应用,适合于临床HFRS血清标本的检测和血清流行病学调查。     

Central European Dobrava Hantavirus isolate from a striped field mouse (Apodemus agrarius)

Dobrava virus (DOBV) is a hantavirus that causes hemorrhagic fever with renal syndrome (HFRS) in Europe. It is hosted by at least two rodent species, Apodemus flavicollis and A. agrarius. According to their natural hosts they form the distinct genetic lineages DOBV-Af and DOBV-Aa, respectively. We have now established a DOBV isolate named Slovakia (SK/Aa) from an A. agrarius animal captured in Slovakia. The complete S and M and partial L segment nucleotide sequences of the new isolate were determined. Phylogenetic analyses showed that the SK/Aa isolate clustered together with the other DOBV-Aa sequences amplified from A. agrarius before and can be taken as the representative of this genetic lineage. SK/Aa, in comparison with a DOBV-Af isolate, was used for serotyping neutralizing antibodies of HFRS patients in Central Europe. Most patients' sera exhibited a higher endpoint titer when probed with our new isolate, suggesting that DOBV-Aa strains are responsible for most of the DOBV-caused HFRS cases in this region.     

Hantavirus disease in Germany due to infection with Dobrava-Belgrade virus genotype Kurkino

Members of the Dobrava-Belgrade virus (DOBV) species are hantaviruses carried by different Apodemus mice as reservoir hosts and causing haemorrhagic fever with renal syndrome (HFRS) in humans. In Central Europe, the Kurkino genotype of DOBV, associated with the striped field mouse, Apodemus agrarius, is prevalent. This paper presents the first extensive study of the serological and molecular diagnostics, epidemiology and clinics of DOBV-Kurkino infections in Central Europe. Serum samples from 570 German patients living in the habitat of A. agrarius (north and northeast Germany) and exhibiting febrile disease, were analysed. All samples were tested by ELISA, subsets of samples were also analysed by immunoblot, neutralization assay, and RT-PCR. A group of 86 individuals was confirmed as DOBV-infected. The virus neutralization assay allowed a reliable identification of DOBV antibodies during both acute and convalescent phases of infection. However, differentiation of relevant DOBV genotypes was not possible by neutralization test but required molecular analysis. Whereas DOBV IgM antibodies tend to persist in the infected organism, RNAaemia seems to be short. Nucleotide sequences were amplified from four patients, and their analysis demonstrated infection by DOBV-Kurkino. With respect to the initial results, the high degree of identity of local patient-derived and A. agrarius-derived virus sequences may allow a closer allocation of the geographical place where the human infection occurred. In contrast to moderate/severe HFRS caused by the DOBV genotypes Dobrava or Sochi, all available data showed a mild clinical course of HFRS caused by DOBV-Kurkino infection without lethal outcomes.