First Molecular Evidence for Puumala Hantavirus in Poland

Puumala virus (PUUV) causes mild to moderate cases of haemorrhagic fever with renal syndrome (HFRS), and is responsible for the majority of hantavirus infections of humans in Fennoscandia, Central and Western Europe. Although there are relatively many PUUV sequences available from different European countries, little is known about the presence of this virus in Poland. During population studies in 2009 a total of 45 bank voles were trapped at three sites in north-eastern Poland, namely islands on Dejguny and Dobskie Lakes and in a forest near Mikołajki. S and M segment-specific RT-PCR assays detected PUUV RNA in three animals from the Mikołajki site. The obtained partial S and M segment sequences demonstrated the highest similarity to the corresponding segments of a PUUV strain from Latvia. Analysis of chest cavity fluid samples by IgG ELISA using a yeast-expressed PUUV nucleocapsid protein resulted in the detection of two seropositive samples, both being also RT-PCR positive. Interestingly, at the trapping site in Mikołajki PUUV-positive bank voles belong to the Carpathian and Eastern genetic lineages within this species. In conclusion, we herein present the first molecular evidence for PUUV in the rodent reservoir from Poland.     

Muju Virus,Harbored by Myodes regulus in Korea,Might Represent a Genetic Variant of Puumala Virus,the Prototype Arvicolid Rodent-Borne Hantavirus

The genome of Muju virus (MUJV), identified originally in the royal vole (Myodes regulus) in Korea, was fully sequenced to ascertain its genetic and phylogenetic relationship with Puumala virus (PUUV), harbored by the bank vole (My. glareolus), and a PUUV-like virus, named Hokkaido virus (HOKV), in the grey red-backed vole (My. rufocanus) in Japan. Whole genome sequence analysis of the 6544-nucleotide large (L), 3652-nucleotide medium (M) and 1831-nucleotide small (S) segments of MUJV, as well as the amino acid sequences of their gene products, indicated that MUJV strains from different capture sites might represent genetic variants of PUUV, the prototype arvicolid rodent-borne hantavirus in Europe. Distinct geographic-specific clustering of MUJV was found in different provinces in Korea, and phylogenetic analyses revealed that MUJV and HOKV share a common ancestry with PUUV. A better understanding of the taxonomic classification and pathogenic potential of MUJV must await its isolation in cell culture.     

Genetic evidence of hantavirus infections in wild rodents from northwestern Colombia

This report builds on recent serological evidence for the presence of hantavirus in northern Colombia by providing sequence-specific and phylogenetic data of hantavirus infections in wild rodents. From August 2007 to August 2008, 354 rodent specimens representing four families were collected in the northwestern Antioquia region of Colombia. Antibodies reactive to Sin Nombre virus and Maciel virus antigens by IgG enzyme-linked immunosorbent assay were found in 15 of 109 (14%) Cherries cane rats (Zygodontomys cherriei), the only sigmodontinae rodents captured. Lung tissue samples from 11 of the 15 seropositive rodents were RT-polymerase chain reaction positive for hantavirus RNA, using primers for the S and M genome segments. Eight of these amplicons were sequenced and phylogenetic analyses indicated RNA of a hantavirus closely related to Calabazo virus, previously found in Panama. This is the first report of the genetic characterization of a hantavirus in rodents in Colombia.     

河南省部分区域肾综合征出血热患者汉坦病毒的分子特征

目的 分析河南省部分区域肾综合征出血热患者汉坦病毒的分子特征.方法 采集2017-2018年就诊于河南省郑州市第六人民医院临床诊断为肾综合征出血热患者血标本32例,采用半巢式PCR筛选汉坦病毒阳性样本,然后再对阳性样本中病毒S片段和M部分片段基因进行扩增和测序,并对得到的S片段基因序列进行组装.所得序列与已分离到的国内...     

The Use of Chimeric Virus-like Particles Harbouring a Segment of Hantavirus Gc Glycoprotein to Generate a Broadly-Reactive Hantavirus-Specific Monoclonal Antibody

Monoclonal antibodies (MAbs) against viral glycoproteins have important diagnostic and therapeutic applications. In most cases, the MAbs specific to viral glycoproteins are raised against intact virus particles. The biosynthesis of viral glycoproteins in heterologous expression systems such as bacteria, yeast, insect or mammalian cells is often problematic due to their low expression level, improper folding and limited stability. To generate MAbs against hantavirus glycoprotein Gc, we have used initially a recombinant yeast-expressed full-length Puumala virus (PUUV) Gc protein. However, this approach was unsuccessful. As an alternative recombinant antigen, chimeric virus-like particles (VLPs) harboring a segment of PUUV Gc glycoprotein were generated in yeast Saccharomyces cerevisiae. A 99 amino acid (aa)-long segment of Gc protein was inserted into the major capsid protein VP1 of hamster polyomavirus at previously defined positions: either site #1 (aa 80–89) or site #4 (aa 280–289). The chimeric proteins were found to self-assemble to VLPs as evidenced by electron microscopy. Chimeric VLPs induced an efficient insert-specific antibody response in immunized mice. Monoclonal antibody (clone #10B8) of IgG isotype specific to hantavirus Gc glycoprotein was generated. It recognized recombinant full-length PUUV Gc glycoprotein both in ELISA and Western blot assay and reacted specifically with hantavirus-infected cells in immunofluorescence assay. Epitope mapping studies revealed the N-terminally located epitope highly conserved among different hantavirus strains. In conclusion, our approach to use chimeric VLPs was proven useful for the generation of virus-reactive MAb against hantavirus Gc glycoprotein. The generated broadly-reactive MAb #10B8 might be useful for various diagnostic applications.     

5株鼠样品中分离汉坦病毒毒株S基因序列比较与分析

目的 了解近年来从宁波口岸捕获的鼠类样品中分离的5个汉坦病毒株遗传学差异。方法 利用RT-PCR方法,对5个汉坦病毒株S基因全长进行扩增、克隆和测序,并将这5个毒株的S基因序列分别与GenBank登录的20个汉坦病毒感毒株进行比较分析。结果 DX1507株和DX1408株S基因核苷酸序列长度为1766 bp与汉城型(SEOV)病毒株 Rn-DH27同源性最为接近,LJ1112株S基因核苷酸序列长度为1772 bp与汉城型(SEOV)病毒株Cherwell同源性最为接近,DX0903株和BL1402株S基因核苷酸序列长度为1725 bp与大别山型(DABV)病毒株 Yongjia-Nc-95和Yongjia-Nc-38同源性最为接近。结论 宁波口岸分离到的汉坦病毒株分别为汉城型和大别山型,对宁波口岸开展汉坦病毒传播防控有重要意义。     

汉坦病毒Z5株M和S片段全基因序列测定及分析

目的测定汉坦病毒Z5株M、S片段的全长序列,了解其分子结构特征,并分析Z5与其他汉坦病毒株的遗传学差异。方法设计引物,用RT-PCR技术扩增Z5株全长M与S片段。PCR产物纯化后克隆入T载体并测定序列及分析。结果Z5株M片段的全基因序列含有3 616个核苷酸,编码1 135个氨基酸。S全基因序列含有1 700个核苷酸,编码429个氨基酸。经核苷酸与氨基酸同源性分析,Z5株应属于汉坦病毒的汉滩型(HTN型)毒株,与Z10株同属一个亚型。结论Z5株病毒和其他汉滩病毒病毒在核苷酸序列上有差异,但在氨基酸水平上的同源性仍较高。     

深圳市肾综合征出血热疫源地宿主动物汉坦病毒分离株的基因分型

目的 对深圳市肾综合征出血热(HFRS)疫源地进行宿主动物汉坦病毒分离,研究分离株的基因分型.方法 采用幼龄长爪沙鼠接种和Vero-E6细胞培养的方法进行汉坦病毒分离,用直接免疫荧光实验进行鉴定.应用型特异性引物进行反转录一套式PCR分别扩增M片段G1、G2区、S片段,并测定核苷酸序列,进行同源性比对和进化树分析.结果 从深圳市褐家鼠肺中成功分离到2株汉坦病毒,命名为SZ2082和SZ2083,经反转录一套式PCR扩增并进行序列测定显示为第2型汉城病毒型(SEOV).通过对3个基因片段序列比较分析发现,SZ2082和SZ2083部分M和S片段核苷酸序列完全一致.M片段G1、G2区核苷酸序列与SEOV国际代表株80-39的同源性分别为96.7%、95.0%,与第1型汉滩病毒型(HTNV)型国际代表株76-118的同源性仅为75.9%、70.3%.S片段与SEOV80-39和HTNV76-118比较,核苷酸同源性分别为95.7%和69.7%,与M片段的结果相似.通过M片段G2区的分析发现,SZ2082与BjFT01、Beijing-Rn、Guang199、HN71-L处于同一分支,同源性为99.0%～99.7%.结论 在深圳市分离到汉坦病毒,通过鉴定其属于汉城型病毒S2亚型.     

Detection of Human Hantavirus Infections in Lithuania

Abstract Background: In Europe certain hantaviruses are known to cause hemorrhagic fever with renal syndrome of different severity. The objective of the present investigation was to study the presence of hantavirus infections in Lithuania. Material and Methods: Two different serum panels from cancer patients (n = 438) and blood donors (n = 299) from Lithuania were tested by monoclonal antibody capture IgG ELISA using yeast-expressed recombinant nucleocapsid (rN) proteins of Puumala virus (PUUV), Hantaan virus (HTNV) and Dobrava virus (DOBV). The reactivity of ELISA-positive sera was proven in Western blot tests using various hantavirus rN proteins. Selected serum samples were further analyzed by focus reduction neutralization assays. Results: In the IgG ELISA 39 sera from the cancer patients and four sera from blood donors were found to be reactive with at least one of the rN proteins. By immunoblot using the three yeast-expressed rN proteins, the ELISA reactivity of 36 of 39 and two of four serum samples from cancer patients and blood donors, respectively, was confirmed; this corresponds to a seroprevalence of 8.2% and 0.7%, respectively. In ELISA, the majority of the samples reacted exclusively with rN proteins of HTNV and DOBV (31 of 36 and one of two in the two groups). In the group of sera selected for serotyping by focus reduction neutralization assay, this dominance was confirmed by the identification of eight DOBV but only four PUUV infections. No infection by HTNV or another hantavirus besides DOBV and PUUV was verified. Anti-hantavirus-positive human sera were detected in all seven investigated counties of Lithuania. Conclusion: In Lithuania at least two hantaviruses, DOBV and PUUV, circulate and cause human infections. Additional investigations are needed to study the seroprevalence more precisely and to search for clinical cases of hantavirus infections.     

汉坦病毒系统发生分析及其基因分型的研究

目的寻找适于汉坦病毒进行系统发生分析的系统发生树构建方法及其基因片段。选用GenBank中部分有代表性的汉坦病毒株，用M、S、M＋S全序列。以及G1、G2的2003—2302、2741—2950核苷酸片段分别以邻位相连法（NJ）和最大简约法（MP）构建系统发生树。用M＋S、M、G1、以及G2的2003—2302片段以NJ法构建的系统发生树将所选25株病毒分为5个分支，分别对应于HTN、SEO、DOB、PUU血清型及引起HPS的Sin Nomhre血清族。但用G2区的2741—2950片段构建的系统发生树将DOB型与HTN型分为一支。以MP法用M、M＋S及G1片段构建的发生树，结果与NJ法相同。但用S与G2的2003—2302、2741—2950片段构建的系统发生树则不能将所有的病毒株分在相应的血清型。因此，汉坦病毒的基因分型及系统发生分析时，NJ法优于MP法；用M片段的G1核苷酸序列可以替代M-9S片段全序列进行基因分型及系统发生分析。     

Genetic Diversity of Puumala orthohantavirus in Rodents and Human Patients in Austria, 2012–2019

Puumala orthohantavirus (PUUV) has a wide distribution throughout Europe. Distinctive temporal patterns of spillover into the human population are related to population dynamics of the reservoir host, the bank vole (Clethrionomys glareolus). As the rodent host is tied to specific habitats with small individual ranges, PUUV genetic diversity is also highly correlated with geographic distance. Using sequenced portions of viral S and M segments, we determined whether geographic clusters were supported. Human cases of PUUV infections are concentrated in southeastern Austria. We detected four distinct genotypes: two genotypes of the Alpe-Adria (ALAD) lineage typically associated with southeast Europe, and two sublineages of the Central Europe (CE) lineage. One cluster of CE genotypes represents a phylogenetically distinct sublineage compared to previously reported CE clades, and extends the boundary of the CE lineage further south than previously reported.     

Characterization of Hantavirus N Protein Intracellular Dynamics and Localization

Hantaviruses are enveloped viruses that possess a tri-segmented, negative-sense RNA genome. The viral S-segment encodes the multifunctional nucleocapsid protein (N), which is involved in genome packaging, intracellular protein transport, immunoregulation, and several other crucial processes during hantavirus infection. In this study, we generated fluorescently tagged N protein constructs derived from Puumalavirus (PUUV), the dominant hantavirus species in Central, Northern, and Eastern Europe. We comprehensively characterized this protein in the rodent cell line CHO-K1, monitoring the dynamics of N protein complex formation and investigating co-localization with host proteins as well as the viral glycoproteins Gc and Gn. We observed formation of large, fibrillar PUUV N protein aggregates, rapidly coalescing from early punctate and spike-like assemblies. Moreover, we found significant spatial correlation of N with vimentin, actin, and P-bodies but not with microtubules. N constructs also co-localized with Gn and Gc albeit not as strongly as the glycoproteins associated with each other. Finally, we assessed oligomerization of N constructs, observing efficient and concentration-dependent multimerization, with complexes comprising more than 10 individual proteins.     

Expression Profile of Human Renal Mesangial Cells Is Altered by Infection with Pathogenic Puumala Orthohantavirus

Acute kidney injury (AKI) with proteinuria is a hallmark of infections with Eurasian orthohantaviruses. Different kidney cells are identified as target cells of hantaviruses. Mesangial cells may play a central role in the pathogenesis of AKI by regulation of inflammatory mediators and signaling cascades. Therefore, we examined the characteristics of hantavirus infection on human renal mesangial cells (HRMCs). Receptor expression and infection with pathogenic Puumala virus (PUUV) and low-pathogenic Tula virus (TULV) were explored. To analyze changes in protein expression in infected mesangial cells, we performed a proteome profiler assay analyzing 38 markers of kidney damage. We compared the proteome profile of in vitro-infected HRMCs with the profile detected in urine samples of 11 patients with acute hantavirus infection. We observed effective productive infection of HRMCs with pathogenic PUUV, but only poor abortive infection for low-pathogenic TULV. PUUV infection resulted in the deregulation of proteases, adhesion proteins, and cytokines associated with renal damage. The urinary proteome profile of hantavirus patients demonstrated also massive changes, which in part correspond to the alterations observed in the in vitro infection of HRMCs. The direct infection of mesangial cells may induce a local environment of signal mediators that contributes to AKI in hantavirus infection.     

Genetic characterization of hantaviruses associated with sigmodontine rodents in an endemic area for hantavirus pulmonary syndrome in southern Brazil

An ecological assessment of reservoir species was conducted in a rural area (Jaborá) in the mid-west of the state of Santa Catarina in southern Brazil, where hantavirus pulmonary syndrome is endemic, to evaluate the prevalence of hantavirus infection in wild rodents. Blood and tissue samples were collected from 507 rodents during seven field trips from March 2004 to April 2006. Some of the animals were karyotyped to confirm morphological identification. Phylogenetic reconstructions of rodent specimens, based on the mitochondrial DNA cytochrome b gene sequences, were also obtained. Hantavirus antibody was found in 22 (4.3%) of the 507 rodents: 5 Akodon montensis, 2 Akodon paranaensis, 14 Oligoryzomys nigripes, and 1 Sooretamys angouya. Viral RNAs detected in O. nigripes and A. montensis were amplified and sequenced. O. nigripes virus genome was 97.5% (nt) and 98.4% (nt) identical to sequences published for Araucaria (Juquitiba-like) virus based on N and G2 fragment sequences. Viral sequences from A. montensis strain showed 89% and 88% nucleotide identities in a 905-nt fragment of the nucleocapsid (N) protein-coding region of the S segment when it was compared with two other Akodontine rodent-associated viruses from Paraguay, A. montensis and Akodon cursor, respectively. Phylogenetic analysis showed the cocirculation of two genetic hantavirus lineages in the state of Santa Catarina, one from O. nigripes and the other from A. montensis, previously characterized in Brazil and Paraguay, respectively. The hantavirus associated with A. montensis, designed Jaborá virus, represents a distinct phylogenetic lineage among the Brazilian hantaviruses.     

Newly recognized hantaviruses associated with hantavirus pulmonary syndrome in northern Brazil: partial genetic characterization of viruses and serologic implication of likely reservoirs

Following the occurrence of the first laboratory-confirmed cases of hantavirus pulmonary syndrome (HPS) in Maranhao State, Brazil, rodents were trapped and rodent materials screened by ELISA for antibodies to Sin Nombre and Andes hantaviruses. Antibody-positive samples were tested by RT-PCR, amplified products were sequenced, and phylogenetic trees were constructed for comparison with known hantaviruses. From 104 rodent blood samples collected (40 Bolomys lasiurus, 52 Holochilus sciureus, 12 Oligoryzomys fornesi, and one Proechimys guyannensis), 21 (20.2%) were antibody-positive (one B. lasiurus, five O. fornesi, and 15 H. sciureus). Hantavirus RNA was amplified by PCR from two O. fornesi and four H. sciureus. Viral sequencing identified two hantavirus genotypes. The genotype recovered from O. fornesi, is designated herein as Anajatuba (ANAJ) and the genotype recovered from H. sciureus is designated Rio Mearim (RIME). Phylogenetic analysis of a 643-nucleotide region of the N segment showed both viruses to be most closely related (94-96% nucleotide homology) to Río Mamoré virus, a virus associated with Oligoryzomys microtis in Bolivia and Peru, but not found in northern Brazil. O. fornesi was frequently captured in and around human dwellings. H. sciureus, is a semi-aquatic rodent captured only in remote areas rarely frequented by humans.     

Innate and adaptive immune responses against human Puumala virus infection: immunopathogenesis and suggestions for novel treatment strategies for severe hantavirus‐associated syndromes

Two related hyperinflammatory syndromes are distinguished following infection of humans with hantaviruses: haemorrhagic fever with renal syndrome (HFRS) seen in Eurasia and hantavirus pulmonary syndrome (HPS) seen in the Americas. Fatality rates are high, up to 10% for HFRS and around 35%–40% for HPS. Puumala virus (PUUV) is the most common HFRS‐causing hantavirus in Europe. Here, we describe recent insights into the generation of innate and adaptive cell‐mediated immune responses following clinical infection with PUUV. First described are studies demonstrating a marked redistribution of peripheral blood mononuclear phagocytes (MNP) to the airways, a process that may underlie local immune activation at the site of primary infection. We then describe observations of an excessive natural killer (NK) cell activation and the persistence of highly elevated numbers of NK cells in peripheral blood following PUUV infection. A similar vigorous CD8 Tcell response is also described, though Tcell responses decline with viraemia. Like MNPs, many NK cells and CD8 T cells also localize to the lung upon acute PUUV infection. Following this, findings demonstrating the ability of hantaviruses, including PUUV, to cause apoptosis resistance in infected target cells, are described. These observations, and associated inflammatory cytokine responses, may provide new insights into HFRS and HPS disease pathogenesis. Based on similarities between inflammatory responses in severe hantavirus infections and other hyperinflammatory disease syndromes, we speculate whether some therapeutic interventions that have been successful in the latter conditions may also be applicable in severe hantavirus infections.     

Identification of Seoul hantavirus in Rattus norvegicus in Indonesia

The first genetic evidence for the presence of Seoul hantavirus (SEOV) in Indonesia is presented. Partial M segment sequence was recovered from the lung tissue of Rattus norvegicus trapped in central Jakarta. The sequence belongs to SEOV genotype and is most closely related to the strain B-1 from Japan.     

Complete Genome and Phylogeny of Puumala Hantavirus Isolates Circulating in France

Puumala virus (PUUV) is the agent of nephropathia epidemica (NE), a mild form of hemorrhagic fever with renal syndrome (HFRS) in Europe. NE incidence presents a high spatial variation throughout France, while the geographical distribution of the wild reservoir of PUUV, the bank vole, is rather continuous. A missing piece of the puzzle is the current distribution and the genetic variation of PUUV in France, which has been overlooked until now and remains poorly understood. During a population survey, from 2008 to 2011, bank voles were trapped in eight different forests of France located in areas known to be endemic for NE or in area from where no NE case has been reported until now. Bank voles were tested for immunoglobulin (Ig)G ELISA serology and two seropositive animals for each of three different areas (Ardennes, Jura and Orleans) were then subjected to laboratory analyses in order to sequence the whole S, M and L segments of PUUV. Phylogenetic analyses revealed that French PUUV isolates globally belong to the central European (CE) lineage although isolates from Ardennes are clearly distinct from those in Jura and Orleans, suggesting a different evolutionary history and origin of PUUV introduction in France. Sequence analyses revealed specific amino acid signatures along the N protein, including in PUUV from the Orleans region from where NE in humans has never been reported. The relevance of these mutations in term of pathophysiology is discussed.