Boris Klempa

Novel Hantavirus Sequences in Shrew, Guinea

To the Editor: Hantaviruses, family Bunyaviridae, have been known as causative agents of hemorrhagic fever with renal syndrome in Asia and Europe (1,2) and hantavirus cardiopulmonary syndrome in the Americas (3). Hantaviruses are spread by aerosolized rodent excreta and are strongly associated with their natural hosts, rodents of the family Muridae. Based on phylogenetic analyses, hantaviruses have been divided into 3 major groups that resemble 3 subfamilies of their natural hosts (Figure, panel A). Figure Maximum likelihood phylogenetic analysis of hantaviruses showing the phylogenetic placement of Tan826 (Tanganya virus, indicated by arrow) based on partial L segment nucleotide (A) and amino acid (B) sequences and partial S segment amino acid sequences ... Recently, we found the first indigenous African hantavirus, Sangassou virus (SANGV), in an African wood mouse (Hylomyscus simus) collected in Guinea (5). Thottapalayam virus (TPMV), isolated from an Asian house shrew (Suncus murinus) in India (6), is the only known hantavirus to be hosted by a shrew instead of a rodent (7,8). We report the recovery of hantavirus RNA of a novel sequence from a shrew, collected in Guinea, West Africa. During a study of rodentborne hemorrhagic fever viruses performed in Guinea in 2002–2004, 32 shrews of the genus Crocidura were collected and screened for hantavirus RNA by reverse transcription–PCR (5). An RNA sample designated Tan826 produced a PCR product of the expected size. The animal host was a male Crocidura theresae collected in the grassland savannah around the village Tanganya (10°00′02″N, 10°58′22″W) in January 2004. Species identification, following the taxonomic nomenclature (9), was performed on the basis of morpho-anatomical characteristics and was supported by molecular analyses. Partial L segment sequence of 412 nt was determined by cloning and sequencing of the obtained PCR product. Nucleotide sequence comparisons between Tan826 and other representatives of the genus Hantavirus showed very low sequence identity values, ranging from 67.7% (Andes virus) to 72.3% (Puumala virus). Corresponding sequences of deduced viral RNA polymerase (137 aa) showed only slightly higher similarity values of 69.3% (Tula virus) to 76.6% (SANGV). In a maximum likelihood phylogenetic tree (Figure, panel A), Tan826 did not unambiguously cluster with any of the major groups (i.e., Murinae-, Arvicolinae-, Sigmodontinae-associated viruses) and showed equal relatedness to all 3 groups. This exceptional position of the Tan826 sequence within the tree is consistent with its detection in a shrew instead of a rodent host. Because the sequence is only distantly related to other hantaviruses, sequences from additional members of the Bunyaviridae family were analyzed. Despite use of a suboptimal dataset of very divergent and short sequences, the phylogenetic placement of Tan862 within the genus Hantavirus could be clearly demonstrated (Figure, panel B). Furthermore, a partial S segment sequence (442 nt, 147 aa of the putative nucleoprotein) was determined to compare Tan826 directly with the shrew-associated TPMV (for which only an S segment sequence was available in GenBank). Rather unexpectedly, the Tan826 sequence showed the lowest similarity to TPMV: 47.5% on nt level and 39.4% on aa level. The identity values to other Hantavirus members were also extremely low, 52.2% (Sin Nombre virus) to 62.1% (SANGV) on nt level and 50.6% (Andes virus) to 56.7% (Hantaan, Dobrava virus) on aa level. Corresponding aa sequences were then used for phylogenetic analysis to reduce problems derived from higher sequence diversities. In the resulting evolutionary tree, Tan826 and TPMV did not join any of the 3 major groups but also did not cluster together (Figure, panel C). Our attempts to obtain more sequence data were hampered by the unique nature of the Tan826 virus sequence, which makes it difficult to design additional effective PCR primers, as well as by the limited amount of available biological material from the shrew. Nevertheless, the sequence and phylogenetic analyses of the 2 partial sequences strongly indicate that they represent a novel hantavirus. The amino acid sequences are highly divergent (≈25%–50%) from those of other hantaviruses and in phylogenetic trees; the Tan826 virus sequence appeared approximately equally related to those of all other hantaviruses. We propose to name the putative new species Tanganya virus (TGNV), after the locality where it was detected. Detecting the virus in 1 of 32 Crocidura shrews, 15 of them C. theresae, is not sufficient to define C. theresae as a reservoir animal of this novel virus. However, the unique position of TGNV in evolutionary trees supports the idea that a shrew instead of a rodent is the natural host of TGNV. Therefore, it is rather surprising that TGNV did not form a monophyletic group with TPMV. Before this observation becomes either a challenge or support for the hantavirus–host coevolution concept, more extensive sequence data (for comprehensive phylogenetic analysis) and epizootiologic studies (to confirm the natural hosts of both viruses) are necessary. TGNV represents, after the recently described SANGV (5), a second hantavirus from Africa. Its low sequence similarity to other hantaviruses should make this virus serologically distinct from other hantaviruses, as shown for TPMV (10). Therefore, human infections by TGNV might be missed when using antibody detection assays based on antigens from conventional hantaviruses.

Hantavirus in bat, Sierra Leone

To the Editor: Hantaviruses (family Bunyaviridae) are transmitted from rodent reservoirs to humans. These viruses cause life-threatening human diseases: hantavirus cardiopulmonary syndrome in the Americas and hemorrhagic fever with renal syndrome in Asia and Europe (1). Since 2006, indigenous hantaviruses were reported also from Africa. Sangassou virus was found in an African wood mouse (Hylomyscus simus) in Guinea (2). Discovery of newer African hantaviruses, Tanganya virus and recently Azagny virus, was even more surprising because they were found in shrews (3,4). The detection of hantaviruses in small mammals other than rodents, such as shrews and also moles (4), increasingly raises questions regarding the real hantavirus host range. Bats (order Chiroptera) are already known to harbor a broad variety of emerging pathogens, including other bunyaviruses (5). Their ability to fly and social life history enable efficient pathogen maintenance, evolution, and spread. Therefore, we conducted a study on hantaviruses in bats from Africa. A total of 525 tissue samples from 417 bats representing 28 genera were tested for the presence of hantavirus RNA. Samples originated from different regions in western and central Africa and were collected during 2009 and early 2011. Total RNA was extracted from tissue samples and reverse transcribed. cDNA was screened by PCR specific for sequences of the large genomic segment across the genus Hantavirus (2). One sample yielded a product of the expected size and was subjected to cloning and sequencing. The positive sample (MGB/1209) was obtained from 1 of 18 investigated slit-faced bats (family Nycteridae). The animal was trapped at the Magboi River within Gola National Park, Sierra Leone (7°50.194′N, 10°38.626′W), and the identification as Nycteris hispida has been verified with the voucher specimen (RCJF529). Histologic examination of organs of the animal showed no obvious pathologic findings. The obtained 414-nt sequence covers a genomic region, which was found to correspond to nt position 2,918–3,332 in the large segment open reading frame of prototypic Hantaan virus. Bioinformatic analysis on the amino acid level showed highest degrees of identity to shrew- and mole-associated hantaviruses (Thottapalayam virus 73.0%, Altai virus 69.7%, Nova and Imjin virus 69.3%). On the basis of tree topology of a maximum-likelihood phylogenetic tree, the sequence does not cluster with rodent-associated hantaviruses but groups with those found in shrews and moles (Figure). Figure Maximum-likelihood phylogenetic tree of MGB/1209 virus based on partial large segment sequence (414 nt) and showing the phylogenetic placement of the novel sequence from Nycteris spp. bat compared with hantaviruses associated (i) with shrews and moles: ... Considering that bats are more closely related to shrews and moles than to rodents (6), a certain genetic similarity of a putative bat-borne hantavirus with shrew- and mole-associated hantaviruses seems reasonable. Notably, shrew-associated Thottapalayam virus (India) and Imjin virus (South Korea) seem to be closer relatives, and African Tanganya virus (Guinea) and Azagny virus (Cote d’Ivoire) are more distantly related. Additional sequence data is needed for more conclusive phylogenetic analyses. Because the new amino acid sequence is at least 22% divergent from those of other hantaviruses, we conclude that the bat was infected with a newly found hantavirus. We propose the putative name Magboi virus (MGBV) for the new virus because it was detected in an animal captured at the Magboi River in Sierra Leone. The MGBV nucleotide sequence is novel and has not been known or handled before in our laboratory. Before this study, hantavirus nucleic acid was found in lung and kidney tissues of bats from the genera Eptesicus and Rhinolophus in South Korea. However, nucleotide sequencing showed the presence of prototypical Hantaan virus indicating a spillover infection or laboratory contamination (7). Further screening is necessary to confirm N. hispida as a natural reservoir host of the virus. Although the presented bat-associated sequence is obviously distinct from other hantaviruses, which suggests association with a novel natural host, a spillover infection from another, yet unrecognized host cannot be ruled out. However, detection of the virus exclusively in 1 organ (lung but not in liver, kidney, and spleen; data not shown) suggests a persistent infection that is typically observed in natural hosts of hantaviruses (8). To date, only a few reports exist on cases of hemorrhagic fever with renal syndrome in Africa (9,10). However, underreporting must be assumed because the symptoms resemble those of many other febrile infections. Moreover, in cases of infections by non–rodent-associated hantaviruses, cross-reactivity with routinely used rodent-borne virus antigens should be limited and may hamper human serodiagnostics (1). The results suggest that bats, which are hosts of many emerging pathogens (5), may act as natural reservoirs for hantavirus. The effect of this virus on public health remains to be determined.

Human pathogenic hantaviruses and prevention of infection

Hantaviruses are emerging viruses which are hosted by small mammals. When transmitted to humans, they can cause two clinical syndromes, hemorrhagic fever with renal syndrome or hantavirus cardiopulmonary syndrome. The review compiles the current list of hantaviruses which are thought to be pathogenic in humans on the basis of molecular or at least serological evidence. Whereas induction of a neutralizing humoral immune response is considered to be protective against infection, the dual role of cellular immunity (protection versus immunopathogenicity) is discussed. For active immunisation, inactivated virus vaccines are licensed in certain Asian countries. Moreover, several classical and molecular vaccine approaches are in pre-clinical stages of development. The development of hantavirus vaccines is hampered by the lack of adequate animal models of hantavirus-associated disease. In addition to active immunization strategies, the review summarizes other ways of infection prevention, as passive immunization, chemoprophylaxis, and exposition prophylaxis.

Hemorrhagic Fever with Renal Syndrome Caused by 2 Lineages of Dobrava Hantavirus, Russia1

These lineages, including a new lineage associated with the novel rodent host Apodemus ponticus, are emerging.

Occurrence of Renal and Pulmonary Syndrome in a Region of Northeast Germany Where Tula Hantavirus Circulates

ABSTRACT Hantavirus species Tula (TULV) is carried by European common voles (Microtus spp.). Its pathogenic potential for humans is unknown. In a rural region of northeast Germany, a 43-year-old man became ill with fever, renal syndrome, and pneumonia. Typing of late acute- and convalescent-phase sera by focus reduction neutralization assay revealed the presence of neutralizing antibodies against TULV. Moreover, we detected TULV genetic material in Microtus arvalis animals that were trapped at places only a few kilometers from the home village of the patient. Phylogenetic analysis of completely sequenced genomic S segments from three virus strains grouped them within a third genetic lineage of the TULV species. This is the first case of hemorrhagic fever with renal syndrome and pulmonary involvement which can be associated with TULV infection.

Genetic interaction between distinct dobrava hantavirus subtypes in Apodemus agrarius and A. flavicollis in nature

ABSTRACT Dobrava virus (DOBV) occurs in two different rodent species, Apodemus flavicollis (DOBV-Af) and A. agrarius (DOBV-Aa). We sequenced the S and M genomic segments from sympatric DOBV-Af and DOBV-Aa strains which fell into two distinct genetic lineages. Molecular phylogenetic analyses gave evidence for genetic reassortment between S and M segments of DOBV-Af and DOBV-Aa and indicated homologous recombination events in DOBV evolution. DOBV-Af and DOBV-Aa are distinct but also subject to genetic exchanges that affect their evolutionary trajectories.

Complex evolution and epidemiology of Dobrava-Belgrade hantavirus: definition of genotypes and their characteristics

Dobrava-Belgrade virus (DOBV) is a human pathogen that has evolved in, and is hosted by, mice of several species of the genus Apodemus. We propose a subdivision of the species Dobrava-Belgrade virus into four related genotypes – Dobrava, Kurkino, Saaremaa, and Sochi – that show characteristic differences in their phylogeny, specific host reservoirs, geographical distribution, and pathogenicity for humans.

Hantaviruses and climate change

Most hantaviruses are rodent-borne emerging viruses. They cause two significant human diseases, haemorrhagic fever with renal syndrome in Asia and Europe, and hantavirus cardiopulmonary syndrome in the Americas. Very recently, several novel hantaviruses with unknown pathogenic potential have been identified in Africa and in a variety of insectivores (shrews and a mole). Because there is very limited information available on the possible impact of climate change on all of these highly dangerous pathogens, it is timely to review this aspect of their epidemiology. It can reasonably be concluded that climate change should influence hantaviruses through impacts on the hantavirus reservoir host populations. We can anticipate changes in the size and frequency of hantavirus outbreaks, the spectrum of hantavirus species and geographical distribution (mediated by changes in population densities), and species composition and geographical distribution of their reservoir hosts. The early effects of global warming have already been observed in different geographical areas of Europe. Elevated average temperatures in West-Central Europe have been associated with more frequent Puumala hantavirus outbreaks, through high seed production (mast year) and high bank vole densities. On the other hand, warm winters in Scandinavia have led to a decline in vole populations as a result of the missing protective snow cover. Additional effects can be caused by increased intensity and frequency of extreme climatic events, or by changes in human behaviour leading to higher risk of human virus exposure. Regardless of the extent of climate change, it is difficult to predict the impact on hantavirus survival, emergence and epidemiology. Nevertheless, hantaviruses will undoubtedly remain a significant public health threat for several decades to come.

Hantaviruses--globally emerging pathogens.

Hantaviruses are emerging zoonotic viruses which cause human disease in Africa, America, Asia, and Europe. This review summarizes the progress in hantavirus epidemiology and diagnostics during the previous decade. Moreover, we discuss the influence of ecological factors on the worldwide virus distribution and give an outlook on research perspectives for the next years.

First Molecular Identification of Human Dobrava Virus Infection in Central Europe

ABSTRACT Viral RNA was amplified by reverse transcription-PCR from a patient suffering from hemorrhagic fever with renal syndrome (HFRS) in Germany. The virus strain could be assigned to the Dobrava hantavirus (DOBV). This is the first molecular identification of human infection by DOBV in central Europe and the first proof that a virus strain related to the DOBV-Aa lineage, carried by Apodemus agrarius rodents, is able to cause HFRS.