Case report: severe neurological manifestation of Dobrava hantavirus infection

In Europe infection with Puumala or Dobrava viruses causes hemorrhagic fever with renal syndrome (HFRS). In the course of HFRS, mild neurological symptoms such as headache, vertigo, and nausea are common. However, the data about the occurrence of severe, potentially life-threatening neurological manifestations are rather scarce. Here, we present a case of HFRS with serologically proven Dobrava virus infection complicated by epileptic seizures and hemiparesis due to focal encephalitis.     

Genetic characterization of new Dobrava hantavirus isolate from Greece

The first complete genome sequence of Dobrava hantavirus isolated from yellow-necked mouse Apodemus flavicollis trapped in the northeastern Greece is described. The S, M, and L segments of the Greek isolate of Dobrava virus are 1673, 3635, and 6532 nucleotides (nt) long, respectively, and encode the nucleocapsid (N) protein of 429 amino acids (aa), glycoprotein precursor of 1135 aa, and the L protein of 2151 aa. N protein contains three cysteine residues conserved in all known hantaviruses, as well as structural domains responsible for the RNA binding and presumable interaction with the apoptosis enhancer Daxx. All cysteine residues and glycosylation sites that are conserved among G1G2 sequences of all hantaviruses species were also found in the Greek isolate. The L protein contains all the polymerase motifs and structural domains found in other hantavirus polymerases. Comparison of the Greek isolate of Dobrava virus with other hantaviruses showed the highest level of sequence homology with Dobrava virus isolate from Slovenia. Other hantaviruses carried by Murinae rodents (Saaremaa, Hantaan, Seoul, and Thailand viruses) were more divergent and hantaviruses carried by Arvicolinae or Sigmodontinae rodents showed the highest genetic diversity with the Greek isolate of Dobrava. The results of phylogenetic analyses confirmed these observations and showed a monophily of all the Dobrava virus strains that, in turn, shared more ancient ancestors first with Saaremaa virus and then with other Murinae-borne hantaviruses.     

Hantavirus infection during a stay in a mountain hut in Northern Slovakia

Hantaviruses in Europe cause human hemorrhagic fever with renal syndrome (HFRS) with various degree of severity. The most severe form is caused by the Dobrava/Belgrade virus (DOBV), associated with the rodent Apodemus flavicollis. During the last decade cases of infection caused by DOBV have been reported in Central Europe. The present study is a report on two Czech patients with severe HFRS who were infected during their stay in a mountain hut in Northern Slovakia. The two patients, combined with a third case observed in the same year in a nearby village in the Czech Republic, suggest that this region in Central Europe has to be considered as endemic for HFRS.     

Genetic characterisation of a Hantavirus isolated from a laboratory-acquired infection

Seoul (SEO) viruses belong to the Hantavirus genus (family Bunyaviridae), cause the moderate form of haemorrhagic fever with renal syndrome, and have been associated with laboratory-acquired infections in many countries. To investigate the pedigree of an isolate, designated IR461, which was obtained from a laboratory-acquired infection in a UK research institute, we determined the nucleotide sequences of the small (S) and medium (M) genome segments. In addition, we determined the sequences of the S segments of two Chinese isolates (R22 and L99) and an American isolate (Tchoupitoulas [TCH]). The S segments range within 1769-1785 nucleotides in length and showed identities of >88% in nucleotide sequence and 97% in amino acid sequence to those of published S segment sequences. The M RNA segment of IR461 is 3651 nucleotides long and shows >84% identity at the nucleotide level and >98% at the amino acid level to the M segments of other SEO viruses. These data confirm that SEO viruses show the least diversity within the Hantavirus genus. Phylogenetic analyses of the sequences showed geographic clustering of the Chinese SEO viruses, and that IR461 was more closely related to SEO viruses isolated in the New World than to those from the Far East.     

Co‐circulation of three pathogenic hantaviruses: Puumala,Dobrava, and Saaremaa in Hungary

Hantaviruses (Bunyaviridae) cause hemorrhagic fever with renal syndrome (HFRS) in Eurasia and hantavirus (cardio)pulmonary syndrome (HCPS) in the Americas. HFRS is caused by Hantaan virus (HTNV), Seoul virus (SEOV), Dobrava virus (DOBV), Saaremaa virus (SAAV), and Puumala virus (PUUV). Of those, only HTNV is not present in Europe. In recent years, hantaviruses, described in other parts of Europe, were also detected at various locations in Hungary. To study the genetic properties of Hungarian hantaviruses in detail, sequences of the viral S and M segments were recovered from bank voles (Myodes glareolus), yellow‐necked mice (Apodemus flavicollis), and striped field mice (Apodemus agrarius) trapped in the Transdanubian region. As expected, the sequences recovered belonged, respectively, to PUUV (two strains), DOBV (one strain), and SAAV (one strain). On phylogenetic trees two new Hungarian PUUV strains located within the well‐ supported Alpe‐Adrian (ALAD) genetic lineage that included also Austrian, Slovenian, and Croatian strains. Analysis of the Hungarian SAAV and DOBV genetic variants showed host‐specific clustering and also geographical clustering within each of these hantavirus species. Hungarian SAAV and DOBV strains were related most closely to strains from Slovenia (Prekmurje region). This study confirms that multiple hantaviruses can co‐circulate in the same locality and can be maintained side‐by‐side in different rodent species. J. Med. Virol. 81:2045–2052, 2009. © 2009 Wiley‐Liss, Inc.     

Hantaviruses in Estonia

Human serum samples collected from healthy individuals in 14 counties were screened by ELISA in order to investigate the presence of hantavirus infections in Estonia. Out of 1,234 serum samples, 124 were found positive for hantavirus-specific IgG and were subsequently serotyped by a focus reduction neutralization test. A total of 112 samples neutralized at least one of the examined hantaviruses-Puumala (PUUV), Saaremaa (SAAV), Dobrava (DOBV), Hantaan, and Seoul viruses-and thereby, the focus reduction neutralization test confirmed the overall hantavirus seroprevalence rate in Estonia to be 9.1%. Most of the sera showed a specific reaction (at least 4-fold higher endpoint titer) of neutralizing antibodies to PUUV (5.1%), while 3.4% showed a SAAV- or SAAV/DOBV-specific reaction. The fact that seven sera (0.6%) could not be serotyped may indicate the presence of an unknown hantavirus serotype. Hantavirus infections were confirmed in 13 of 14 investigated counties, with highly varying seroprevalence rates (1.0-28.4%). The sex ratio was 1.8:1.0 (M:F), and the antibody prevalence peaked in the age group 45-54 years. A total of 513 rodents of seven species trapped in seven counties were examined for the presence of hantavirus antigen, in order to study the distribution of hantavirus natural carriers. Two species, Clethrionomys glareolus and Apodemus agrarius, were found positive for hantaviral antigen in 13.7% and 4.5% of the investigated rodents, respectively. Analyses of viral sequences recovered from infected C. glareolus tissue samples showed that the infecting virus belonged to the PUUV genotype, confirming that PUUV circulates in mainland Estonia. The Estonian PUUV strains were placed in the closest proximity to Russian PUUV strains in phylogenetic trees, suggesting a common evolutionary history. Together with earlier data on SAAV in A. agrarius, the results revealed that two hantaviruses, PUUV and SAAV, are common in Estonia and that the incidence of human infection is high in both cases.     

Hantavirus cardiopulmonary syndrome due to Puumala virus in Germany

In Germany Puumala virus (PUUV), known to cause mild forms of hemorrhagic fever with renal syndrome (HFRS), is the predominating endemic hantavirus. We herein describe an unusually severe case of a PUUV infection that occurred in summer 2015 in South Eastern Germany in a region known to be endemic for PUUV since over ten years. A 54-year-old female gardener was admitted to hospital with fever, cough and dyspnea. Within 48 hours the patient developed a rapid progressive adult respiratory distress syndrome (ARDS) with circulatory failure and required ECMO (extracorporeal membrane oxygenation) treatment. Serological and molecular biological examinations of serum samples confirmed an infection with PUUV. Partial sequences of the S- and M-segment clustered to a strain previously described in South Eastern Germany. Our reported case highlights, that in rare incidents PUUV can cause hantavirus cardiopulmonary syndrome, a syndrome that is usually found after infections with New World hantaviruses, and neurological symptoms.     

Rodent host specificity of European hantaviruses: evidence of Puumala virus interspecific spillover

In order to investigate rodent host specificity of European hantaviruses, experimental infection of colonized and wild-trapped rodents was performed. In addition to the natural rodent reservoir, Clethrionomys glareolus, Puumala hantavirus (PUUV) could infect colonized Microtus agrestis and Lemmus sibiricus, but not Syrian hamsters or Balb/C mice. Neither C. glareolus, nor M. agrestis, could be readily infected by Tula hantavirus (TULV). Wild-trapped Apodemus flavicollis and A. agrarius, the natural reservoirs of Dobrava (DOBV) and Saaremaa (SAAV) hantaviruses, respectively, could both be infected by SAAV. NMRI mice could also be infected by SAAV, but with lower efficiency as compared to Apodemus mice. Balb/C and NMRI laboratory mice, but not C. glareolus, could be infected by DOBV. To our knowledge, this is the first time DOBV and SAAV have been shown to infect adult laboratory mice. Moreover, potential hantavirus spillover infections were investigated in wild-trapped rodents. In addition to the natural host C. glareolus, we also found M. arvalis and A. sylvaticus with a history of PUUV infection. We did not find any C. glareolus or A. sylvaticus infected with TULV, a hantavirus which is known to circulate in the same geographical regions of Belgium.     

Genetic detection of Dobrava/Belgrade virus in a Czech patient with Haemorrhagic fever with renal syndrome

In the summer of 2008, a 15-year-old boy was hospitalized in a paediatric intensive care unit in the Czech Republic. Laboratory diagnosis of hantavirus infection was established by serological and molecular methods. Sequence and phylogenetic analyses showed that the causative strain was Dobrava/Belgrade virus, which is genetically closer to strains associated with Apodemus flavicollis rodents.     

汉坦病毒浙江分离株ZT71株的全基因核苷酸序列测定及分析

目的 为了获得浙江省汉坦病毒基因组更为详尽的资料,研究汉坦病毒的进化状况及变异程度,为疫苗病毒株的选择使用提供科学依据.方法 设计特异性引物,RT-PCR分段扩增ZT71株全长L、M和S片段,PCR产物纯化后克隆于T载体并进行序列测定.然后进行遗传进化分析.结果 汉坦病毒ZT71株L、M及S基因分别由6530、3651和1753个核苷酸组成,分别编码2151、1133和429个氨基酸.基因比较分析表明,ZT71株与SEO型汉坦病毒比较其L片段核苷酸同源性为95.5%～99.7%,氨基酸同源性为99.0%～99.3%;M片段核苷酸同源性为84.1%～99.6%,氨基酸同源性为95.3%～99.2%;S片段核苷酸同源性为88.7%～99.5%,氨基酸同源性为96.5%～99.1%;而与其他汉坦病毒同源性则较低.结论 测定了ZT71株的全基因核苷酸序列,其L、M和S片段具有和其他汉坦病毒L、M、S片段相似的核苷酸一级结构,但与SEO型更为接近,属于SEO型病毒.     

Puumala and Dobrava viruses cause hemorrhagic fever with renal syndrome in Bosnia-Herzegovina: Evidence of highly cross-neutralizing antibody responses in early patient sera

Hantavirus infection was diagnosed serologically by μ-capture IgM and IgG ELISAs in hemorrhagic fever with renal syndrome (HFRS) patients admitted to Tuzla Hospital, Bosnia-Herzegovina. The results indicated that more than one hantavirus caused the outbreak. To address the question of which hantavirus serotypes were involved, sequentially drawn sera were analyzed by focus reduction neutralization test (FRNT) for antibodies against Puumala, Hantaan, Dobrava, and Seoul hantaviruses. The data revealed that acute- or early convalescent-phase sera, even when drawn as late as 3 weeks after the onset of disease, could not be used for typing of the causative hantavirus; a significant number of these samples showed similar reactivity of neutralizing antibodies to several different hantavirus serotypes. Moreover, although several acute-phase sera showed the highest FRNT titer to Hantaan virus, convalescent sera from these patients in all cases showed high specificity for Puumala or Dobrava viruses. This phenomenon, interpreted as a cross-neutralizing primary antibody response, makes several earlier reports concerning causative agents of HFRS questionable. Serological examination of small rodents trapped in the endemic area identified Puumala- and Dobrava-like virus infections. RT-PCR and sequencing of rodent lung samples identified Dobrava virus in one yellow-necked field mouse (Apodemus flavicollis). Cross-FRNT data, using polyclonal rabbit antibodies, clearly confirmed Dobrava virus as a unique hantavirus serotype. In conclusion, the results revealed that both Puumala- and Dobrava-like viruses caused HFRS in Bosnia-Herzegovina, whereas no signs of Hantaan or Seoul virus involvement were found. J. Med. Virol. 53:51–59, 1997. © 1997 Wiley-Liss, Inc.     

Hantavirus infection in Iranian patients suspected to viral hemorrhagic fever

Background: Hantaviruses are a group of emerging pathogens causing hemorrhagic fever with renal syndrome and Hantavirus cardiopulmonary syndrome in human. This study was conducted to investigate Hantavirus infection among Iranian viral hemorrhagic fever suspected patients. Methods: From April 2014 to June 2016, 113 cases from 25 different provinces of Iran were analyzed for Hantavirus infection by IgM/IgG ELISA and pan-Hantavirus RT-PCR tests. Results: Although, viral genome was detected in none of the subjects, IgM and IgG antibodies were detected in 19 and 4 cases, respectively. Differentiation of the anti-Hantavirus antibodies according to virus species by EUROLINE Anti-Hantavirus Profile Kit revealed three Puumala virus IgM positive, one Hantaan virus IgM positive, one Hantaan virus IgM borderline, and two Puumala virus IgG borderline cases. Conclusions: This study demonstrates the circulation of Hantaviruses in Iran and calls for further investigations of these life-threatening viruses in the country.     

汉坦病毒感染的量子点标记免疫检测研究

目的 应用量子点荧光标记技术和间接免疫法,建立一种新的汉坦病毒（HV）感染的免疫检测技术.方法 采用碳二亚胺交联法,在水溶性量子点表面修饰protein G和羊抗人IgG.以HV抗原片为固相载体,QDs-PG-IgG复合物为标记二抗,检测待检血清中的HV特异性抗体,并对检测条件进行优化.结果 量子点与羊抗人IgG的最佳偶联条件：反应时间2h、pH 6.0、羊抗人IgG浓度20 μg/ml.检测体系的最佳反应条件：量子点标记二抗的最适工作浓度为1：200,HV感染阳性血清检测的最大稀释度为1：1280.结论 成功建立了简便、快速的HV感染的量子点标记免疫检测方法,该方法具有良好的灵敏度和特异性,抗荧光猝灭性强,为进一步实现HV抗体的单分子定量检测奠定基础.     

Clinical and laboratory findings in patients with oliguric and non-oliguric Hantavirus haemorrhagic fever with renal syndrome: an analysis of 128 patients

Patients with haemorrhagic fever with renal syndrome (HFRS) may present without significant oliguria. We compared different initial clinical symptoms and laboratory findings in patients who developed oliguric acute renal failure (ARF) with those in patients who did not develop oliguric ARF. Overall, 128 patients with serologically confirmed HFRS were hospitalized at the University Hospital for Infectious Disease, Zagreb, Croatia between January 1999 and December 2010. Clinical signs and laboratory findings were extracted from medical charts, and were assessed for their relationship to the development of oliguric ARF. Puumala virus infection was diagnosed in 101 (79%) patients, and Dobrava-Belgrade virus infection in 27 (21%). Oliguria or anuria developed in 30% of patients. We identified the following risk factors for the development of oliguria and anuria on multivariable analysis: conjunctival hyperaemia or bleeding (relative risk (RR) 1.84, 95% CI 1.09–3.10; p 0.023), diarrhoea (RR 1.45, 95% CI 1.07–1.97; p 0.017), serum sodium of ≤133 mM (RR 2.21, 95% CI 1.34–3.64; p 0.002), and dipstick protein value of >1.5 g/L (RR 1.59, 95% CI 1.09–2.33; p 0.016), as well as hiking in the forest (RR 1.92, 95% CI 1.13–3.26; p 0.016). Our findings may help physicians in the earlier identification of patients with a more severe form of HFRS caused by Puumala and Dobrava-Belgrade viruses. Particular attention should be given to findings such as conjunctival hyperaemia or bleeding, diarrhoea, a low serum sodium level, and proteinuria.     

Dobrava virus infection: serological diagnosis and cross-reactions to other hantaviruses

Recent data have shown that Dobrava (DOB) hantavirus is the cause of severe haemorrhagic fever with renal syndrome (HFRS) in central and eastern Europe. To determine whether serological assays need to be based on the homologous viral antigen rather than on closely related hantavirus antigens, acute and convalescent sera from patients with HFRS collected in former Yugoslavia were examined for IgM and IgG to three hantavirus antigens; DOB, Hantaan (HTN) and Puumala (PUU). Focus reduction neutralization test was included for comparison and confirmation of the enzyme-linked immunosorbent assay (ELISA) results. Although the results showed that the cross-reactivity was high between these three antigens during the acute phase of the disease, one of 155 patients serum samples reacted only in the DOB antigen-based IgM assay. The evaluation of IgG reactivities revealed that a DOB antigen-based IgG ELISA has to be used in sero-epidemiological studies; 7.1% (11/155) of the acute phase/early convalescent sera and 12.5% (2/16) of the late convalescent sera, respectively, reacted only with the homologous DOB antigen.     

呼吸道合胞病毒感染的研究进展

呼吸道合胞病毒是在世界范围内引起婴幼儿呼吸道感染的最常见病原微生物,它不仅可以引起呼吸系统的一些常见症状和体征,而且在肺外器官如中枢神经系统、心血管系统、内分泌系统等各器官也可引起一些尚未被人们普遍认识的肺外表现,而这些临床特点的重要性也愈来愈受到人们的重视.     

深圳市HFRS疫源地鼠携带汉坦病毒与人感染汉坦病毒的基因特征对应性研究

目的 了解深圳市肾综合征出血热(HFRS)疫源地鼠携带汉坦病毒与人感染汉坦病毒二者的病原学特征及联系,为HFRS的防控提供科学依据.方法 收集疑似HFRS病人急性期血清标本,并在相同区域开展笼日法捕鼠,无菌取鼠肺.分别采用ELISA和直接免疫荧光法筛选阳性标本,选择代表性血清标本以及鼠肺标本进行逆转录-套式聚合酶链反应(RT-nested PCR)扩增部分M和S片段及核苷酸序列测定,与国内外的汉坦病毒毒株一起进行同源性比对和系统进化树分析.结果 472份鼠肺经直接免疫荧光检测共获得阳性鼠肺47份,带毒率为9.96%.对60份IgM抗体阳性的血液标本进行RT-nested PCR扩增,检出12份阳性,阳性率为20%.从代表性的8份鼠肺和8份病人血清中提取的标本二者之间M片段核苷酸序列的同源性高达95%以上,S片段之间的同源性高达96.5%以上.其推导的氨基酸序列同源性分别为98.0%～100%、98.4%～100%.深圳市HFRS疫源地鼠携带的汉坦病毒和人感染汉坦病毒均为汉城型S2亚型.结论 深圳市流行的汉坦病毒为SEO型S2亚型.无论是同一地区的鼠和人之间,还是不同地区的鼠和人之间,汉坦病毒都是具有较高的同源性.     

Genetic evidence for the presence of two distinct hantaviruses associated with Apodemus mice in Croatia and analysis of local strains

In Europe, Dobrava-Belgrade (DOBV), Saaremaa (SAAV), and Puumala (PUUV) viruses are known to cause hemorrhagic fever with renal syndrome (HFRS). All three hantaviruses are now found in Croatia. Lung tissue samples of 315 Apodemus mice trapped in 2003-2004 were screened for the presence of hantaviral N-Ag and 20 mice (6.3%) were found either strongly positive or weak/suspected-positive. Partial sequences of hantavirus M and S segments were recovered by RT-PCR from six mice and subjected to (phylo)genetic analysis that revealed the presence of four novel strains of DOBV and one of SAAV. Curiously, one of the newly described DOBV strains was found in Apodemus agrarius mouse, that is, not in the traditional host, A. flavicollis mice, suggesting a spillover event. S segment sequences recovered previously from HFRS cases [Markoti? et al., 2002] were confirmed as DOBV sequences; one of which appeared particularly close to the prototype Slovenian DOBV isolate. Taken together with earlier data on PUUV in Croatia, these results show a co-circulation of three European hantavirus pathogens in this country. So far, not a single SAAV sequence has been recovered from HFRS patients either in Croatia or neighboring Slovenia and Hungary nor in Slovakia suggesting a somewhat lower fequency of acute SAAV infection in humans in this part of Europe than for example in the Baltics.     

热休克蛋白70与汉坦病毒持续性感染乳大鼠大脑皮层星形胶质细胞

目的 探讨热休克蛋白70（HSP70）过表达可能有助于汉坦病毒（HTNV）持续感染。 方法 在HTNV持续性感染过程中,通过Westen blotting及RT-PCR检测大脑皮层星形胶质细胞中HSP70的表达情况,并通过免疫荧光显微技术、AnnexinV 及RT-PCR检测细胞凋亡。 结果 与对照组相比,感染HTNV 76-118或 SEOV L99 的星形胶质细胞HSP70蛋白的表达增加,且在HTNV 76-118感染后3d或SEOV L99感染后2d HSP70蛋白表达达到峰值（n=15,P＜0.01,P＜0.05）;感染HTNV 76-118及SEOV L99上调星形胶质细胞HSP70基因的表达,且在HTNV 76-118感染后2~5d或SEOV L99感染后2~4d HSP70基因表达达到峰值（n=15,P＜0.01,P＜0.05）。HTNV76-118或SEOV L99感染过程中,星形胶质细胞无细胞病变效应,并且病毒复制未诱导细胞凋亡。 结论 HTNV感染星形胶质细胞激活了HSP70,这种改变可能抑制细胞凋亡,促进病毒持续性感染。