Selection of viral variants during persistent infection of insectivorous bat cells with Middle East respiratory syndrome coronavirus.

Coronaviruses that cause severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) are speculated to have originated in bats. The mechanisms by which these viruses are maintained in individuals or populations of reservoir bats remain an enigma. Mathematical models have predicted long-term persistent infection with low levels of periodic shedding as a likely route for virus maintenance and spillover from bats. In this study, we tested the hypothesis that bat cells and MERS coronavirus (CoV) can co-exist in vitro. To test our hypothesis, we established a long-term coronavirus infection model of bat cells that are persistently infected with MERS-CoV. We infected cells from Eptesicus fuscus with MERS-CoV and maintained them in culture for at least 126 days. We characterized the persistently infected cells by detecting virus particles, protein and transcripts. Basal levels of type I interferon in the long-term infected bat cells were higher, relative to uninfected cells, and disrupting the interferon response in persistently infected bat cells increased virus replication. By sequencing the whole genome of MERS-CoV from persistently infected bat cells, we identified that bat cells repeatedly selected for viral variants that contained mutations in the viral open reading frame 5 (ORF5) protein. Furthermore, bat cells that were persistently infected with ΔORF5 MERS-CoV were resistant to superinfection by wildtype virus, likely due to reduced levels of the virus receptor, dipeptidyl peptidase 4 (DPP4) and higher basal levels of interferon in these cells. In summary, our study provides evidence for a model of coronavirus persistence in bats, along with the establishment of a unique persistently infected cell culture model to study MERS-CoV-bat interactions.

Genetic diversity and ecology of coronaviruses hosted by cave-dwelling bats in Gabon.

Little research on coronaviruses has been conducted on wild animals in Africa. Here, we screened a wide range of wild animals collected in six provinces and five caves of Gabon between 2009 and 2015. We collected a total of 1867 animal samples (cave-dwelling bats, rodents, non-human primates and other wild animals). We explored the diversity of CoVs and determined the factors driving the infection of CoVs in wild animals. Based on a nested reverse transcription-polymerase chain reaction, only bats, belonging to the Hipposideros gigas (4/156), Hipposideros cf. ruber (13/262) and Miniopterus inflatus (1/249) species, were found infected with CoVs. We identified alphacoronaviruses in H. gigas and H. cf. ruber and betacoronaviruses in H. gigas. All Alphacoronavirus sequences grouped with Human coronavirus 229E (HCoV-229E). Ecological analyses revealed that CoV infection was significantly found in July and October in H. gigas and in October and November in H. cf ruber. The prevalence in the Faucon cave was significantly higher. Our findings suggest that insectivorous bats harbor potentially zoonotic CoVs; highlight a probable seasonality of the infection in cave-dwelling bats from the North-East of Gabon and pointed to an association between the disturbance of the bats' habitat by human activities and CoV infection.

Widespread arenavirus occurrence and seroprevalence in small mammals, Nigeria.

BACKGROUND: Lassa fever, killing thousands of people annually, is the most reported viral zoonotic disease in Nigeria. Recently, different rodent species carrying diverse lineages of the Lassa virus (LASV) in addition to a novel Mobala-like genetic sequence were detected within the country. Here, screening 906 small mammal specimens from 11 localities for IgG antibodies and incorporating previous PCR detection data involving the same populations, we further describe arenavirus prevalence across Nigeria in relation to host species and geographical location. METHODS: Small mammals were trapped during the period 2011-2015 according to geographical location (endemic and non-endemic zones for Lassa fever), season (rainy and dry seasons between 2011 and 2012 for certain localities) and habitat (indoors, peridomestic settings and sylvatic vegetation). Identification of animal specimens from genera such as Mastomys and Mus (Nannomys) was assisted by DNA sequencing. Small mammals were tested for LASV IgG antibody using an indirect immunofluorescence assay (IFA). RESULTS: Small mammals were infected in both the endemic and non-endemic zones for Lassa fever, with a wider range of species IgG-positive (n = 8) than those which had been previously detected to be PCR-positive (n = 3). IgG-positive species, according to number of infected individuals, were Mastomys natalensis (n = 40), Mastomys erythroleucus (n = 15), Praomys daltoni (n = 6), Mus baoulei (n = 5), Rattus rattus (n = 2), Crocidura spp. (n = 2), Mus minutoides (n = 1) and Praomys misonnei (n = 1). Multimammate mice (Mastomys natalensis and M. erythroleucus) were the most ubiquitously infected, with animals testing positive by either PCR or IgG in 7 out of the 11 localities sampled. IgG prevalence in M. natalensis ranged from 1% in Abagboro, 17-36 % in Eguare Egoro, Ekpoma and Ngel Nyaki, up to 52 % in Mayo Ranewo. Prevalence according to locality, season and age was not, however, statistically significant for M. natalensis in Eguare Egoro and Ekpoma, localities that were sampled longitudinally. CONCLUSIONS: Overall, our study demonstrates that arenavirus occurrence is probably more widely distributed geographically and in extent of host taxa than is currently realized. This expanded scope should be taken into consideration in Lassa fever control efforts. Further sampling should also be carried out to isolate and characterize potential arenaviruses present in small mammal populations we found to be seropositive.

Geographical distribution and relative risk of Anjozorobe virus (Thailand orthohantavirus) infection in black rats (Rattus rattus) in Madagascar.

BACKGROUND: Hantavirus infection is a zoonotic disease that is associated with hemorrhagic fever with renal syndrome and cardiopulmonary syndrome in human. Anjozorobe virus, a representative virus of Thailand orthohantavirus (THAIV), was recently discovered from rodents in Anjozorobe-Angavo forest in Madagascar. To assess the circulation of hantavirus at the national level, we carried out a survey of small terrestrial mammals from representative regions of the island and identified environmental factors associated with hantavirus infection. As we were ultimately interested in the potential for human exposure, we focused our research in the peridomestic area. METHODS: Sampling was achieved in twenty districts of Madagascar, with a rural and urban zone in each district. Animals were trapped from a range of habitats and examined for hantavirus RNA by nested RT-PCR. We also investigated the relationship between hantavirus infection probability in rats and possible risk factors by using Generalized Linear Mixed Models. RESULTS: Overall, 1242 specimens from seven species were collected (Rattus rattus, Rattus norvegicus, Mus musculus, Suncus murinus, Setifer setosus, Tenrec ecaudatus, Hemicentetes semispinosus). Overall, 12.4% (111/897) of Rattus rattus and 1.6% (2/125) of Mus musculus were tested positive for THAIV. Rats captured within houses were less likely to be infected than rats captured in other habitats, whilst rats from sites characterized by high precipitation and relatively low seasonality were more likely to be infected than those from other areas. Older animals were more likely to be infected, with infection probability showing a strong increase with weight. CONCLUSIONS: We report widespread distribution of THAIV in the peridomestic rats of Madagascar, with highest prevalence for those living in humid areas. Although the potential risk of infection to human may also be widespread, our results provide a first indication of specific zone with high transmission. Gathered data will be helpful to implement policies for control and prevention of human risk infection.

Genetic variants of Cao Bang hantavirus in the Chinese mole shrew (Anourosorex squamipes) and Taiwanese mole shrew (Anourosorex yamashinai).

To determine the genetic diversity and geographic distribution of Cao Bang virus (CBNV) and to ascertain the existence of CBNV-related hantaviruses, natural history collections of archival tissues from Chinese mole shrews (Anourosorex squamipes) and Taiwanese mole shrews (Anourosorex yamashinai), captured in Guizho Province, People's Republic of China, and in Nantou County, Taiwan, in 2006 and 1989, respectively, were analyzed for hantavirus RNA by RT-PCR. Pair-wise alignment and comparison of the S-, M- and L-segment sequences indicated CBNV in two of five Chinese mole shrews and a previously unrecognized hantavirus, named Xinyi virus (XYIV), in seven of 15 Taiwanese mole shrews. XYIV was closely related to CBNV in Vietnam and China, as well as to Lianghe virus (LHEV), recently reported as a distinct hantavirus species in Chinese mole shrews from Yunnan Province in China. Phylogenetic analyses, using maximum-likelihood and Bayesian methods, showed that XYIV shared a common ancestry with CBNV and LHEV, in keeping with the evolutionary relationship between Anourosorex mole shrews. Until such time that tissue culture isolates of CBNV, LHEV and XYIV can be fully analyzed, XYIV and LHEV should be regarded as genetic variants, or genotypes, of CBNV.

Hantaviruses in Finnish soricomorphs: evidence for two distinct hantaviruses carried by Sorex araneus suggesting ancient host-switch.

Hantaviruses are emerging viruses carried by rodents, soricomorphs (shrews and moles) and bats. In Finland, Puumala virus (PUUV) was for years the only hantavirus detected. In 2009, however, Seewis virus (SWSV) was reported from archival common shrew (Sorex araneus) samples collected in 1982 in Finland. To elucidate the diversity of hantaviruses in soricomorphs in Finland, 180 individuals were screened, representing seven species captured from 2001 to 2012: hantavirus RNA was screened using RT-PCR, and hantaviral antigen using immunoblotting with polyclonal antibodies raised against truncated SWSV nucleocapsid protein. The overall hantavirus RNA prevalence was 14% (26/180), antigen could be demonstrated in 9 of 20 SWSV RT-PCR positive common shrews. Genetic analyses revealed that four soricomorph-borne hantaviruses circulate in Finland, including Boginia virus (BOGV) in water shrew (Neomys fodiens) and Asikkala virus (ASIV) in pygmy shrew (Sorex minutus). Interestingly, on two study sites, common shrews harbored strains of two different hantaviruses: Seewis virus and a new distinct, genetically distant (identity 57% at amino acid level) virus (Altai-like virus) which clusters together with viruses in the basal phylogroup I of hantaviruses with 62-67% identity at amino acid level. This is the first evidence of coexistence of two clearly distinct hantavirus species circulating simultaneously in one host species population. The findings suggest an ancient host-switching event from a yet unknown host to S. araneus. In addition, phylogenetic analyses of partial S and M segment sequences showed that SWSV in Finland represents a unique genotype in Europe.

A new hantavirus from the stripe-backed shrew (Sorex cylindricauda) in the People's Republic of China.

Inspired by the recent discovery of genetically distinct hantaviruses from insectivore species worldwide, we performed a small-scale search for insectivore-borne hantaviruses. In this paper, we report the discovery of a new hantavirus, which was designated the Qian Hu Shan virus (QHSV). This virus was detected in the lung tissues of three stripe-backed shrews (Sorex cylindricauda), which were captured in the Yunnan Province, China. The full-length S genomic segment of the representative QHSV strain YN05-284 was 1661 nucleotides and is predicted to encode a nucleocapsid protein of 429 amino acids that starts at nucleotide position 48. It exhibited the highest similarity with other Sorex-related hantaviruses, with 68.1%-72.8% nucleotide and 71.9%-84.4% amino acid sequence identities. An analysis of a 1430-nucleotide region of the partial M segment exhibited approximately 54.4%-79.5% nucleotide and 43.2%-90.8% amino acid sequence identities to other hantaviruses. A comparison of a 432-nucleotide region of the L segment also showed similar degrees of identity, with 68.9%-78.4% nucleotide and 71.1%-93.8% amino acid sequence identities to other hantaviruses. Phylogenetic analyses using Bayesian methods indicated that QHSV shared the most recent common ancestor with other Sorex-related hantaviruses. The host was identified using a morphological assessment and verified using mitochondrial cytochrome b (mt-Cyt b) gene sequencing. A pair-wise comparison of the 1140-nucleotide mt-Cyt b gene sequence from the host demonstrated that the host was close to S. cylindricauda from Nepal with 94.3% identity. The virus-host association tanglegram, which was constructed using the Dendroscope software, indicated that the QHSV phylogeny and the host phylogeny were approximately matched, which suggests no evidence of host switching for QHSV. Our results contribute to a wider viewpoint regarding the heterogeneity of viruses that infect shrews.

Hantaviruses in Africa.

This paper summarizes the progress in the search for hantaviruses and hantavirus infections in Africa. After having collected molecular evidence of an indigenous African hantavirus in 2006, an intensive investigation for new hantaviruses has been started in small mammals. Various novel hantaviruses have been molecularly identified not only in rodents but also in shrews and bats. In addition, the first African hantavirus, Sangassou virus, has been isolated and functionally characterized in cell culture. Less is known about the ability of these hantaviruses to infect humans and to cause diseases. To date, no hantavirus genetic material could be amplified from patients' specimens collected in Africa. Serological studies in West Africa, based on a battery of screening and confirmatory assays, led to the detection of hantavirus antibodies in the human population and in patients with putative hantavirus disease. In addition to this overview, we present original data from seroepidemiological and field studies conducted in the Southern part of Africa. A human seroprevalence rate of 1.0% (n=1442) was detected in the South African Cape Region whereas no molecular evidence for the presence of hantavirus was found in 2500 small animals trapped in South Africa and Namibia.

HFRS and hantaviruses in the Balkans/South-East Europe.

Hemorrhagic fever with renal syndrome is endemic in the Balkans with epidemic outbreaks and sporadic cases that have been recorded yearly since the disease was first recognized. The incidence of Balkan HFRS is modest, with approximately one hundred cases reported in most years. Seroepidemiological investigations conducted in several Balkan countries revealed an overall seroprevalence of 6% in Bosnia and Herzegovina, 1.6% in Croatia, 4% in Greece and 1.7% in Slovenia, respectively. The complex ecology of the Balkan Peninsula supports the existence of diverse rodent and insectivore species which harbor several pathogenic and non-pathogenic hantaviruses. Among them only Dobrava (DOBV) and Puumala (PUUV) viruses are associated with disease in humans. Comprehensive clinical studies compared clinical signs and symptoms between patients infected with either virus. A spectrum of clinical picture of the disease ranges from mild illness typical of PUUV infections to a severe form with fulminant hemorrhagic fever and an overall mortality rate of 9.8% among DOBV infected patients. While severe DOBV cases are recognized from Slovenia in the North to Greece in the South, PUUV infections are more frequent in northern part of the area. Balkans represent an area with a potential need for hantavirus vaccines, but due to co-existence of DOBV and PUUV causing HFRS in the same region, a universal vaccine is required.

High prevalence of Nova hantavirus infection in the European mole (Talpa europaea) in France.

SUMMARY: Recent discovery of genetically distinct hantaviruses in shrews and moles (order Soricomorpha, family Soricidae and Talpidae) has challenged the conventional view that rodents serve as the principal reservoir hosts. Nova virus (NVAV), previously identified in archival liver tissue of a single European mole (Talpa europaea) from Hungary, represents one of the most highly divergent hantaviruses identified to date. To ascertain the spatial distribution and genetic diversity of NVAV, we employed RT-PCR to analyse lungs from 94 moles, captured in two locations in France, during October 2012 to March 2013. NVAV was detected in more than 60% of moles at each location, suggesting efficient enzootic virus transmission and confirming that this mole species serves as the reservoir host. Although the pathogenic potential of NVAV is unknown, the widespread geographical distribution of the European mole might pose a hantavirus exposure risk for humans.

Boginia virus, a newfound hantavirus harbored by the Eurasian water shrew (Neomys fodiens) in Poland.

BACKGROUND: Guided by decades-old reports of hantaviral antigens in the Eurasian common shrew (Sorex araneus) and the Eurasian water shrew (Neomys fodiens) in European Russia, we employed RT-PCR to analyze lung tissues of soricine shrews, captured in Boginia, Huta Dlutowska and Kurowice in central Poland during September 2010, 2011 and 2012. FINDINGS: In addition to Seewis virus (SWSV), which had been previously found in Eurasian common shrews elsewhere in Europe, a genetically distinct hantavirus, designated Boginia virus (BOGV), was detected in Eurasian water shrews captured in each of the three villages. Phylogenetic analysis, using maximum likelihood and Bayesian methods, showed that BOGV formed a separate lineage distantly related to SWSV. CONCLUSIONS: Although the pathogenic potential of BOGV and other recently identified shrew-borne hantaviruses is still unknown, clinicians should be vigilant for unusual febrile diseases and clinical syndromes occurring among individuals reporting exposures to shrews.

Phylogeny and origins of hantaviruses harbored by bats, insectivores, and rodents.

Hantaviruses are among the most important zoonotic pathogens of humans and the subject of heightened global attention. Despite the importance of hantaviruses for public health, there is no consensus on their evolutionary history and especially the frequency of virus-host co-divergence versus cross-species virus transmission. Documenting the extent of hantavirus biodiversity, and particularly their range of mammalian hosts, is critical to resolving this issue. Here, we describe four novel hantaviruses (Huangpi virus, Lianghe virus, Longquan virus, and Yakeshi virus) sampled from bats and shrews in China, and which are distinct from other known hantaviruses. Huangpi virus was found in Pipistrellus abramus, Lianghe virus in Anourosorex squamipes, Longquan virus in Rhinolophus affinis, Rhinolophus sinicus, and Rhinolophus monoceros, and Yakeshi virus in Sorex isodon, respectively. A phylogenetic analysis of the available diversity of hantaviruses reveals the existence of four phylogroups that infect a range of mammalian hosts, as well as the occurrence of ancient reassortment events between the phylogroups. Notably, the phylogenetic histories of the viruses are not always congruent with those of their hosts, suggesting that cross-species transmission has played a major role during hantavirus evolution and at all taxonomic levels, although we also noted some evidence for virus-host co-divergence. Our phylogenetic analysis also suggests that hantaviruses might have first appeared in Chiroptera (bats) or Soricomorpha (moles and shrews), before emerging in rodent species. Overall, these data indicate that bats are likely to be important natural reservoir hosts of hantaviruses.

Hantaan virus surveillance in small mammals at firing points 10 and 60, Yeoncheon, Gyeonggi Province, Republic of Korea.

We used epidemiological data and indirect fluorescent antibody tests to determine the Hantaan virus (HTNV) antibody-positive (Ab+) prevalence in small mammals captured at firing point 10 (FP-10) and firing point 60 (FP-60), Gyeonggi Province, near the demilitarized zone, Republic of Korea (ROK), from 2001 to 2005. We used these data, combined with the partial M segment amplified from HTNV recovered from lung tissues of Apodemus agrarius, to clarify the genetic diversity and phylogenetic relationships among HTNV strains in the ROK. Of the eight species of rodents and one insectivore species captured, A. agrarius accounted for 93.4% and 88.5% at FP-10 and FP-60, respectively. Only two species of rodents, A. agrarius and Micromys minutus, were HTNV Ab+. The overall HTNV Ab+ prevalence for A. agrarius captured at FP-10 and FP-60 was 23.3% (121/520) and 14.5% (94/647), respectively. The hantaviral reverse transcription-polymerase chain reaction-positive rate of Ab+ A. agrarius was 74.2% (167/215), and the phylogenetic trees, based on the 269-nucleotide G2-encoding M segment, demonstrated that HTNV strains from FP-10 and FP-60 were distantly segregated from HTNV of other geographic regions in Korea and China. These data are useful in the development of risk reduction strategies for the prevention of hantavirus infections among military personnel, especially during training or the event of hostilities, and civilian populations.

Molecular evolution of Azagny virus, a newfound hantavirus harbored by the West African pygmy shrew (Crocidura obscurior) in Côte d'Ivoire.

BACKGROUND: Tanganya virus (TGNV), the only shrew-associated hantavirus reported to date from sub-Saharan Africa, is harbored by the Therese's shrew (Crocidura theresae), and is phylogenetically distinct from Thottapalayam virus (TPMV) in the Asian house shrew (Suncus murinus) and Imjin virus (MJNV) in the Ussuri white-toothed shrew (Crocidura lasiura). The existence of myriad soricid-borne hantaviruses in Eurasia and North America would predict the presence of additional hantaviruses in sub-Saharan Africa, where multiple shrew lineages have evolved and diversified. METHODS: Lung tissues, collected in RNAlater®, from 39 Buettikofer's shrews (Crocidura buettikoferi), 5 Jouvenet's shrews (Crocidura jouvenetae), 9 West African pygmy shrews (Crocidura obscurior) and 21 African giant shrews (Crocidura olivieri) captured in Côte d'Ivoire during 2009, were systematically examined for hantavirus RNA by RT-PCR. RESULTS: A genetically distinct hantavirus, designated Azagny virus (AZGV), was detected in the West African pygmy shrew. Phylogenetic analysis of the S, M and L segments, using maximum-likelihood and Bayesian methods, under the GTR+I+Γ model of evolution, showed that AZGV shared a common ancestry with TGNV and was more closely related to hantaviruses harbored by soricine shrews than to TPMV and MJNV. That is, AZGV in the West African pygmy shrew, like TGNV in the Therese's shrew, did not form a monophyletic group with TPMV and MJNV, which were deeply divergent and basal to other rodent- and soricomorph-borne hantaviruses. Ancestral distributions of each hantavirus lineage, reconstructed using Mesquite 2.74, suggested that the common ancestor of all hantaviruses was most likely of Eurasian, not African, origin. CONCLUSIONS: Genome-wide analysis of many more hantaviruses from sub-Saharan Africa are required to better understand how the biogeographic origin and radiation of African shrews might have contributed to, or have resulted from, the evolution of hantaviruses.

Genetic diversity of Imjin virus in the Ussuri white-toothed shrew (Crocidura lasiura) in the Republic of Korea, 2004-2010.

Recently, Imjin virus (MJNV), a genetically distinct hantavirus, was isolated from lung tissues of the Ussuri white-toothed shrew (Crocidura lasiura) captured near the demilitarized zone in the Republic of Korea. To clarify the genetic diversity of MJNV, partial M- and L-segment sequences were amplified from lung tissues of 12 of 37 (32.4%) anti-MJNV IgG antibody-positive Ussuri white-toothed shrews captured between 2004 and 2010. A 531-nucleotide region of the M segment (coordinates 2,255 to 2,785) revealed that the 12 MJNV strains differed by 0-12.2% and 0-2.3% at the nucleotide and amino acid levels, respectively. A similar degree of nucleotide (0.2-11.9%) and amino acid (0-3.8%) difference was found in a 632-nucleotide length of the L segment (coordinates 962 to 1,593) of nine MJNV strains. Phylogenetic analyses, based on the partial M and L segments of MJNV strains generated by the neighbor-joining and maximum likelihood methods, showed geographic-specific clustering, akin to the phylogeography of rodent-borne hantaviruses.

Epizootiological study of tick-borne encephalitis virus infection in Japan.

Tick-borne encephalitis virus (TBEV) is a zoonotic agent causing severe encephalitis in humans. Rodent species that are potential hosts for TBEV are widely distributed in various regions in Japan. In this study, we carried out large-scale epizootiological surveys in rodents from various areas of Japan. A total of 931 rodent and insectivore sera were collected from field surveys. Rodents seropositive for TBEV were found in Shimane Prefecture in Honshu and in several areas of Hokkaido Prefecture. These results emphasize the need for further epizootiological and epidemiological research of TBEV and preventive measures for emerging tick-borne encephalitis in Japan.

Hantavirus evolution in relation to its rodent and insectivore hosts: no evidence for codivergence.

Hantaviruses are considered one of the best examples of a long-term association between RNA viruses and their hosts. Based on the appearance of strong host specificity, it has been suggested that hantaviruses cospeciated with the rodents and insectivores they infect since these mammals last shared a common ancestor, approximately 100 million years ago. We tested this hypothesis of host-virus codivergence in two ways: 1) we used cophylogenetic reconciliation analysis to assess the fit of the virus tree onto that of the host and 2) we estimated the evolutionary rates and divergence times for the Hantavirus genus using a Bayesian Markov Chain Monte Carlo method and similarly compared these with those of their hosts. Our reconciliation analysis provided no evidence for a history of codivergence between hantaviruses and their hosts. Further, the divergence times for the Hantavirus genus were many orders of magnitude too recent to correspond with the timescale of their hosts' speciation. We therefore propose that apparent similarities between the phylogenies of hantaviruses and their mammalian hosts are the result of a more recent history of preferential host switching and local adaptation. Based on the presence of clade-defining amino acids in all genomic segments, we propose that the patterns of amino acid replacement in these viruses are also compatible with a history of host-specific adaptation.

How to diagnose hantavirus infections and detect them in rodents and insectivores.

Hantaviruses are carried by rodents and insectivores in which they cause persistent and generally asymptomatic infections. Several hantaviruses can infect humans and many of them cause either haemorrhagic fever with renal syndrome (HFRS) in Eurasia or hantavirus cardiopulmonary syndrome (HCPS) in the Americas. In humans hantavirus infections are diagnosed using IgM-capture tests but also by RT-PCR detection of viral RNA. For detection of hantavirus infections in rodents and insectivores, serology followed by immunoblotting of, for example, lung tissue, and RT-PCR detection of viral RNA may be used, and if of interest followed by sequencing and virus isolation. For sero/genotyping of hantavirus infections in humans and carrier animals neutralisation tests/RNA sequencing are required. Hantaviruses are prime examples of emerging and re-emerging infections and it seems likely that many new hantaviruses will be detected in the near future.

Development of serological assays for Thottapalayam virus, an insectivore-borne Hantavirus.

Thottapalayam virus (TPMV), a member of the genus Hantavirus in the family Bunyaviridae, was isolated from an insectivore, Suncus murinus (musk shrew), captured in southern India in 1964. While the isolation of TPMV predates the discovery of the prototype Hantaan virus, little is known about its genetics and biology. To date, preliminary evidence suggests that TPMV differs significantly, both antigenically and genetically, from all known rodent-borne hantaviruses. However, since detailed epizootiological studies have not been conducted, it is unclear if TPMV is naturally harbored by an insectivore host or if TPMV represents a "spillover" from its natural rodent reservoir host. Moreover, to what extent TPMV causes infection and/or disease in humans is not known. To address these issues, we first studied the antigenic profile of TPMV using monoclonal antibodies against Hantaan and Seoul viruses and polyclonal immune sera against Puumala virus and TPMV. Armed with this newfound information, we developed an enzyme-linked immunosorbent assay system for the diagnosis of TPMV infections in shrews and humans, using a recombinant TPMV N antigen manipulated to have an E5/G6 epitope to be captured by monoclonal antibody clone E5/G6. Using this assay, we found anti-TPMV antibodies in sera from a patient with high fever of unknown etiology in Thailand and from two shrews captured in Indonesia. Seropositivity was verified by the indirect immunofluorescence antibody test, Western blotting analysis, and focus reduction neutralization test. Collectively, our data indicate that TPMV is harbored by Suncus murinus as its host in nature and is capable of infecting humans.

[The role of small terrestrial mammals in the epidemiology of rabies]. / Uloha drobných zemních savcu v epidemiologii vztekliny.

The examination should help to elucidate the possibility of virus occurrence in free living small terrestrial rodents. The examination was oriented on the following: 1. Animals living in natural surroundings. 2. Rodents examined after injuring man. The examination was carried out by means of the direct immunofluorescent test and, partially, also by biological assay on suckling laboratory mice. In the first part of experiment, more than 10,000 small terrestrial mammals were entrapped, belonging to 16 species. In the second part of experiment, 1,969 rodents, belonging to 12 species, were examined after injuring humans. In these cases, a biological test was also carried out to demonstrate the occurrence of the virus. In neither of the above mentioned experiment the occurrence of rabies was proved.