Elisabeth Fichet-Calvet

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.

Epizootic emergence of Usutu virus in wild and captive birds in Germany

This study aimed to identify the causative agent of mass mortality in wild and captive birds in southwest Germany and to gather insights into the phylogenetic relationship and spatial distribution of the pathogen. Since June 2011, 223 dead birds were collected and tested for the presence of viral pathogens. Usutu virus (USUV) RNA was detected by real-time RT-PCR in 86 birds representing 6 species. The virus was isolated in cell culture from the heart of 18 Blackbirds (Turdus merula). USUV-specific antigen was demonstrated by immunohistochemistry in brain, heart, liver, and lung of infected Blackbirds. The complete polyprotein coding sequence was obtained by deep sequencing of liver and spleen samples of a dead Blackbird from Mannheim (BH65/11-02-03). Phylogenetic analysis of the German USUV strain BH65/11-02-03 revealed a close relationship with strain Vienna that caused mass mortality among birds in Austria in 2001. Wild birds from lowland river valleys in southwest Germany were mainly affected by USUV, but also birds kept in aviaries. Our data suggest that after the initial detection of USUV in German mosquitoes in 2010, the virus spread in 2011 and caused epizootics among wild and captive birds in southwest Germany. The data also indicate an increased risk of USUV infections in humans in Germany.

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.

Current Molecular Epidemiology of Lassa Virus in Nigeria

ABSTRACT Recent Lassa virus strains from Nigeria were completely or partially sequenced. Phylogenetic analysis revealed the predominance of lineage II and III strains, the existence of a previously undescribed (sub)lineage in Nigeria, and the directional spread of virus in the southern part of the country. The Bayesian analysis also provided estimates for divergence times within the Lassa virus clade.

Reproductive characteristics of Mastomys natalensis and Lassa virus prevalence in Guinea, West Africa.

We recently reported increased prevalence of Lassa virus (LASV) infection in Mastomys natalensis during the rainy season in Guinea, West Africa. Here, the association of LASV prevalence with fecundity, fertility, and the age structure of the rodent population was analyzed using data from the previous study. The animals reproduced throughout the year, but highest fecundity was observed during the rainy season. Accordingly, the rodent population was aged at the beginning and young at the end of the rainy season. Lassa virus infection was observed in all age groups, including the very young, which is compatible with vertical transmission. However, since the prevalence of infection showed a trend with increasing age in the younger age groups, horizontal transmission by yet unknown mechanisms may also play a role in LASV transmission. Multivariate analysis did not show an association of LASV prevalence with any demographic variable studied. Rodent behavior influencing virus transmissibility and contaminated environment may therefore be responsible for spatial and seasonal variations in LASV prevalence in M. natalensis.

Using modelling to disentangle the relative contributions of zoonotic and anthroponotic transmission: the case of lassa fever.

Background Zoonotic infections, which transmit from animals to humans, form the majority of new human pathogens. Following zoonotic transmission, the pathogen may already have, or may acquire, the ability to transmit from human to human. With infections such as Lassa fever (LF), an often fatal, rodent-borne, hemorrhagic fever common in areas of West Africa, rodent-to-rodent, rodent-to-human, human-to-human and even human-to-rodent transmission patterns are possible. Indeed, large hospital-related outbreaks have been reported. Estimating the proportion of transmission due to human-to-human routes and related patterns (e.g. existence of super-spreaders), in these scenarios is challenging, but essential for planned interventions. Methodology/Principal Findings Here, we make use of an innovative modeling approach to analyze data from published outbreaks and the number of LF hospitalized patients to Kenema Government Hospital in Sierra Leone to estimate the likely contribution of human-to-human transmission. The analyses show that almost of the cases at KGH are secondary cases arising from human-to-human transmission. However, we found much of this transmission is associated with a disproportionally large impact of a few individuals (‘super-spreaders’), as we found only of human cases result in an effective reproduction number (i.e. the average number of secondary cases per infectious case) , with a maximum value up to . Conclusions/Significance This work explains the discrepancy between the sizes of reported LF outbreaks and a clinical perception that human-to-human transmission is low. Future assessment of risks of LF and infection control guidelines should take into account the potentially large impact of super-spreaders in human-to-human transmission. Our work highlights several neglected topics in LF research, the occurrence and nature of super-spreading events and aspects of social behavior in transmission and detection.

New Hosts of The Lassa Virus.

Lassa virus (LASV) causes a deadly haemorrhagic fever in humans, killing several thousand people in West Africa annually. For 40 years, the Natal multimammate rat, Mastomys natalensis, has been assumed to be the sole host of LASV. We found evidence that LASV is also hosted by other rodent species: the African wood mouse Hylomyscus pamfi in Nigeria, and the Guinea multimammate mouse Mastomys erythroleucus in both Nigeria and Guinea. Virus strains from these animals were isolated in the BSL-4 laboratory and fully sequenced. Phylogenetic analyses of viral genes coding for glycoprotein, nucleoprotein, polymerase and matrix protein show that Lassa strains detected in M. erythroleucus belong to lineages III and IV. The strain from H. pamfi clusters close to lineage I (for S gene) and between II & III (for L gene). Discovery of new rodent hosts has implications for LASV evolution and its spread into new areas within West Africa.

Seroepidemiological study reveals regional co-occurrence of Lassa- and Hantavirus antibodies in Upper Guinea, West Africa

To assess the public health relevance of Lassa arenavirus and hantavirus infections in a subpopulation of recently febrile patients.

Lassa serology in natural populations of rodents and horizontal transmission.

Lassa virus causes hemorrhagic fever in West Africa. Previously, we demonstrated by PCR screening that only the multimammate mouse, Mastomys natalensis, hosts Lassa virus in Guinea. In the present study, we used the same specimen collection from 17 villages in Coastal, Upper, and Forest Guinea to investigate the Lassa virus serology in the rodent population. The aim was to determine the dynamics of antibody development in M. natalensis and to detect potential spillover infections in other rodent species. Immunoglobulin G (IgG) antibody screening was performed using the indirect immunofluorescence assay with the Guinean Lassa virus strain Bantou 289 as antigen. The overall seroprevalence was 8% (129/1551) with the following rodents testing positive: 109 M. natalensis, seven Mastomys erythroleucus, four Lemniscomys striatus, four Praomys daltoni, three Mus minutoides, and two Praomys rostratus. Nearly all of them (122/129) originated from Bantou, Tanganya, and Gbetaya, where Lassa virus is highly endemic in M. natalensis. The antibody seroprevalence in M. natalensis from this high-endemic area (27%; 108/396) depended on the village, habitat, host age, and host abundance. A main positive factor was age; the maximum seroprevalence reached 50% in older animals. Our data fit with a model implicating that most M. natalensis rodents become horizontally infected, clear the virus within a period significantly shorter than their life span, and develop antibodies. In addition, the detection of antibodies in other species trapped in the habitats of M. natalensis suggests spillover infections.

A Unified Framework for the Infection Dynamics of Zoonotic Spillover and Spread

A considerable amount of disease is transmitted from animals to humans and many of these zoonoses are neglected tropical diseases. As outbreaks of SARS, avian influenza and Ebola have demonstrated, however, zoonotic diseases are serious threats to global public health and are not just problems confined to remote regions. There are two fundamental, and poorly studied, stages of zoonotic disease emergence: ‘spillover’, i.e. transmission of pathogens from animals to humans, and ‘stuttering transmission’, i.e. when limited human-to-human infections occur, leading to self-limiting chains of transmission. We developed a transparent, theoretical framework, based on a generalization of Poisson processes with memory of past human infections, that unifies these stages. Once we have quantified pathogen dynamics in the reservoir, with some knowledge of the mechanism of contact, the approach provides a tool to estimate the likelihood of spillover events. Comparisons with independent agent-based models demonstrates the ability of the framework to correctly estimate the relative contributions of human-to-human vs animal transmission. As an illustrative example, we applied our model to Lassa fever, a rodent-borne, viral haemorrhagic disease common in West Africa, for which data on human outbreaks were available. The approach developed here is general and applicable to a range of zoonoses. This kind of methodology is of crucial importance for the scientific, medical and public health communities working at the interface between animal and human diseases to assess the risk associated with the disease and to plan intervention and appropriate control measures. The Lassa case study revealed important knowledge gaps, and opportunities, arising from limited knowledge of the temporal patterns in reporting, abundance of and infection prevalence in, the host reservoir.