Brita Auste

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.

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.

Hantavirus Disease Outbreak in Germany: Limitations of Routine Serological Diagnostics and Clustering of Virus Sequences of Human and Rodent Origin

ABSTRACT In Europe, hemorrhagic fever with renal syndrome results mainly from infection with Puumala virus (PUUV) or Dobrava virus. For 31 patients from a hantavirus disease outbreak in Lower Bavaria, a district in southeast Germany, serodiagnosis was undertaken by enzyme-linked immunosorbent assay, immunofluorescence assay, and immunoblot analysis. In a few of these cases, however, PUUV-specific typing of antibodies by these standard assays failed and a virus neutralization assay under biosafety level 3 conditions was required to verify the infection by this virus type. PUUV RNA was amplified by reverse transcription-PCR from acute-phase sera of three patients and was found to be very closely related to virus sequences obtained from bank voles (Clethrionomys glareolus) trapped in the same area. These findings link the outbreak with a novel PUUV lineage, “Bavaria,” circulating in the local rodent population. The Bavaria lineage associated with the outbreak is only distantly related to other PUUV lineages from Germany.

Dobrava-belgrade virus spillover infections, Germany.

We present the molecular identification of Apodemus agrarius (striped field mouse) as reservoir host of the Dobrava-Belgrade virus (DOBV) lineage DOBV-Aa in 3 federal states of Germany. Phylogenetic analyses provided evidence for multiple spillover of DOBV-Aa to A. flavicollis, a crucial prerequisite for host switch and genetic reassortment.

Serological evidence of human hantavirus infections in Guinea, West Africa.

We recently discovered a novel hantavirus, Sangassou virus, in Guinea, West Africa. Using enzyme-linked immunosorbent assays followed by confirmatory and serotyping assays, we retrospectively detected hantavirus antibodies in 3 (4.4%) of 68 patients with fever of unknown origin in Sangassou village, Forest Guinea. A population-based survey in Forest Guinea (n = 649) found the prevalence of hantavirus antibodies to be 1.2%. Specific neutralizing antibodies against Sangassou virus were demonstrated in serum samples from 2 patients and in 2 serum samples of the serosurvey. Our data allow us to conclude that hantavirus infections may be a significant unrecognized medical problem in at least this part of Africa.

Sangassou virus, the first hantavirus isolate from Africa, displays genetic and functional properties distinct from those of other murinae-associated hantaviruses.

ABSTRACT We have discovered the first indigenous African hantavirus, Sangassou virus (SANGV). The virus was isolated from an African wood mouse (Hylomyscus simus), trapped in a forest habitat in Guinea, West Africa. Here, we report on the characterization of the genetic and functional properties of the virus. The complete genome of SANGV was determined and showed typical hantavirus organization. The small (S), medium (M), and large (L) genome segments containing genes encoding nucleocapsid protein, two envelope glycoproteins, and viral polymerase were found to be 1,746, 3,650, and 6,531 nucleotides long, respectively. The exact 5′ and 3′ termini for all three segments of the SANGV genome were determined and were predicted to form the panhandle structures typical of bunyaviruses. Phylogenetic analyses of all three segment sequences confirmed SANGV as a Murinae-associated hantavirus most closely related to the European Dobrava-Belgrade virus. We showed, however, that SANGV uses β1 integrin rather than β3 integrin and decay-accelerating factor (DAF)/CD55 as an entry receptor. In addition, we demonstrated a strong induction of type III lambda interferon (IFN-λ) expression in type I IFN-deficient Vero E6 cells by SANGV. These properties are unique within Murinae-associated hantaviruses and make the virus useful in comparative studies focusing on hantavirus pathogenesis.

A Novel Cardiotropic Murine Adenovirus Representing a Distinct Species of Mastadenoviruses

ABSTRACT During cell culture isolation experiments to recover Dobrava hantavirus from a suspension of liver from a striped field mouse (Apodemus agrarius), an unknown virus was coisolated. Atypically for hantaviruses, it had extensive cytopathic effects. Using a random PCR approach, it was identified as a novel murine adenovirus, MAdV-3 (for MAdV type 3). A plaque-purified virus clone was prepared and further characterized. The complete genome sequence of MAdV-3 was determined to be 30,570 bp in length. Sequence comparisons to other adenovirus species revealed highest similarity to MAdV-1, the representative of the murine adenovirus A species. However, substantial differences were found in the E1, E3, and E4 genomic regions. The phylogenetic distance of MAdV-3 amino acid sequences for pVIII, protease, polymerase, and hexon from MAdV-1 is markedly higher than 0.1 exchange per position, and, based on our cross-neutralization experiments, MAdV-3 and MAdV-1 can be regarded as different serotypes. Therefore, we propose to classify MAdV-3 as the first isolate of a novel adenovirus species, designated murine adenovirus C (MAdV-C). The novel MAdV-3 virus is not only genetically and serologically distinct from MAdV-1 but also shows a unique organ tropism in infected mice. In contrast to MAdV-1, the virus was not detectable in brain but predominantly infected heart tissue. Thus, infection of mice with cardiotropic MAdV-3 might be an interesting animal model of adenovirus-induced myocarditis.

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

ABSTRACT 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.

Isolation of Sochi Virus From a Fatal Case of Hantavirus Disease With Fulminant Clinical Course

Sochi virus, a novel genetic variant of Dobrava-Belgrade virus, was isolated in cell culture from a fulminant lethal case of hantavirus disease presenting with shock and combined kidney and lung failure. Sochi virus is transmitted to humans from host reservoir Apodemus ponticus and must be considered a life-threatening emerging agent.