Genetic characterization of new Dobrava hantavirus isolate from Greece

The first complete genome sequence of Dobrava hantavirus isolated from yellow-necked mouse Apodemus flavicollis trapped in the northeastern Greece is described. The S, M, and L segments of the Greek isolate of Dobrava virus are 1673, 3635, and 6532 nucleotides (nt) long, respectively, and encode the nucleocapsid (N) protein of 429 amino acids (aa), glycoprotein precursor of 1135 aa, and the L protein of 2151 aa. N protein contains three cysteine residues conserved in all known hantaviruses, as well as structural domains responsible for the RNA binding and presumable interaction with the apoptosis enhancer Daxx. All cysteine residues and glycosylation sites that are conserved among G1G2 sequences of all hantaviruses species were also found in the Greek isolate. The L protein contains all the polymerase motifs and structural domains found in other hantavirus polymerases. Comparison of the Greek isolate of Dobrava virus with other hantaviruses showed the highest level of sequence homology with Dobrava virus isolate from Slovenia. Other hantaviruses carried by Murinae rodents (Saaremaa, Hantaan, Seoul, and Thailand viruses) were more divergent and hantaviruses carried by Arvicolinae or Sigmodontinae rodents showed the highest genetic diversity with the Greek isolate of Dobrava. The results of phylogenetic analyses confirmed these observations and showed a monophily of all the Dobrava virus strains that, in turn, shared more ancient ancestors first with Saaremaa virus and then with other Murinae-borne hantaviruses.     

Puumala and Dobrava viruses cause hemorrhagic fever with renal syndrome in Bosnia-Herzegovina: Evidence of highly cross-neutralizing antibody responses in early patient sera

Hantavirus infection was diagnosed serologically by μ-capture IgM and IgG ELISAs in hemorrhagic fever with renal syndrome (HFRS) patients admitted to Tuzla Hospital, Bosnia-Herzegovina. The results indicated that more than one hantavirus caused the outbreak. To address the question of which hantavirus serotypes were involved, sequentially drawn sera were analyzed by focus reduction neutralization test (FRNT) for antibodies against Puumala, Hantaan, Dobrava, and Seoul hantaviruses. The data revealed that acute- or early convalescent-phase sera, even when drawn as late as 3 weeks after the onset of disease, could not be used for typing of the causative hantavirus; a significant number of these samples showed similar reactivity of neutralizing antibodies to several different hantavirus serotypes. Moreover, although several acute-phase sera showed the highest FRNT titer to Hantaan virus, convalescent sera from these patients in all cases showed high specificity for Puumala or Dobrava viruses. This phenomenon, interpreted as a cross-neutralizing primary antibody response, makes several earlier reports concerning causative agents of HFRS questionable. Serological examination of small rodents trapped in the endemic area identified Puumala- and Dobrava-like virus infections. RT-PCR and sequencing of rodent lung samples identified Dobrava virus in one yellow-necked field mouse (Apodemus flavicollis). Cross-FRNT data, using polyclonal rabbit antibodies, clearly confirmed Dobrava virus as a unique hantavirus serotype. In conclusion, the results revealed that both Puumala- and Dobrava-like viruses caused HFRS in Bosnia-Herzegovina, whereas no signs of Hantaan or Seoul virus involvement were found. J. Med. Virol. 53:51–59, 1997. © 1997 Wiley-Liss, Inc.     

Genetic characterization of seoul hantavirus originated from norway rats (Rattus norvegicus) captured in Belgium

Hantaviruses (genus Hantavirus, family Bunyaviridae) cause hemorrhagic fever with renal syndrome (HFRS) in Eurasia and hantavirus (cardio)pulmonary syndrome (HCPS) in the Americas. So far, in Europe, four pathogenic hantaviruses have been found, often in co-circulation: Puumala virus (PUUV), Dobrava virus (DOBV), Saaremaa virus (SAAV), and Seoul virus (SEOV). Of those, only PUUV was found in Belgium. Recently, in our search for hantaviruses in the Flanders region of Belgium we collected genetic and serological evidence for the presence of SEOV virus in local brown rats. In this article, the results of (phylo)genetic analysis of wild-type SEOV strain from the Flanders are presented. The analysis based on the complete S segment sequence and also partial M- and L-segment sequences revealed that the Belgian SEOV strain was related most closely to strains from France, Indonesia, Japan, Singapore, Cambodia (those associated with the species Rattus norvegicus) and Vietnam. Such a clustering was in perfect agreement with the results of direct sequence comparison and suggested the same evolutionary history for all three genome segments of the Belgian SEOV strain (i.e., no reassortment of genome segments). So far, SEOV has been found in two European countries, France and Belgium, and there is every reason to believe that the area of the virus distribution in Europe is not restricted to those countries.     

Co‐circulation of three pathogenic hantaviruses: Puumala,Dobrava, and Saaremaa in Hungary

Hantaviruses (Bunyaviridae) cause hemorrhagic fever with renal syndrome (HFRS) in Eurasia and hantavirus (cardio)pulmonary syndrome (HCPS) in the Americas. HFRS is caused by Hantaan virus (HTNV), Seoul virus (SEOV), Dobrava virus (DOBV), Saaremaa virus (SAAV), and Puumala virus (PUUV). Of those, only HTNV is not present in Europe. In recent years, hantaviruses, described in other parts of Europe, were also detected at various locations in Hungary. To study the genetic properties of Hungarian hantaviruses in detail, sequences of the viral S and M segments were recovered from bank voles (Myodes glareolus), yellow‐necked mice (Apodemus flavicollis), and striped field mice (Apodemus agrarius) trapped in the Transdanubian region. As expected, the sequences recovered belonged, respectively, to PUUV (two strains), DOBV (one strain), and SAAV (one strain). On phylogenetic trees two new Hungarian PUUV strains located within the well‐ supported Alpe‐Adrian (ALAD) genetic lineage that included also Austrian, Slovenian, and Croatian strains. Analysis of the Hungarian SAAV and DOBV genetic variants showed host‐specific clustering and also geographical clustering within each of these hantavirus species. Hungarian SAAV and DOBV strains were related most closely to strains from Slovenia (Prekmurje region). This study confirms that multiple hantaviruses can co‐circulate in the same locality and can be maintained side‐by‐side in different rodent species. J. Med. Virol. 81:2045–2052, 2009. © 2009 Wiley‐Liss, Inc.     

Fatal illness associated with a new hantavirus in Louisiana

A fatal case of hantaviral illness occurred in Louisiana, outside of the range of P. maniculatus, the rodent reservoir for Sin Nombre virus. Hantavirus RNA and antigens were detected in patient autopsy tissues, and nucleotide sequence analysis of amplified polymerase chain reaction (PCR) products identified a newly recognized unique hantavirus, provisionally named Bayou virus. Prominent features of the clinical illness are compatible with hantavirus pulmonary syndrome (HPS), but several features such as renal insufficiency and intraalveolar hemorrhage are more compatible with hemorrhagic fever with renal syndrome (HFRS), a disease associated with Eurasian hantaviruses. © 1995 Wiley-Liss, Inc.     

Diagnostic rapid tests for acute hantavirus infections: specific tests for Hantaan,Dobrava and Puumala viruses versus a hantavirus combination test

Hantaviruses infecting humans in Eurasia include Hantaan, Seoul, Puumala and the closely related Dobrava and Saaremaa viruses. These viruses are causative agents of hemorrhagic fever with renal syndrome (HFRS), which is recognized as a severe health care problem in several countries. Diagnostics of hantavirus infections relies on serology, performed principally with enzyme immunoassay (EIA) or immunofluorescence assay (IFA). We developed four 5-min immunochromatographic IgM-antibody tests for diagnostics of acute Puumala, Dobrava and Hantaan virus infections and a similar combination test to detect all Eurasian pathogenic hantavirus infections. We evaluated the assays using 100 fingertip blood samples collected randomly from Finnish volunteers, 28 confirmed hantavirus IgM-negative sera, and 77 sera from patients with acute infections of various hantaviruses. The specificities and sensitivities of the Puumala-, Dobrava- and Hantaan virus -specific tests varied from 96 to 100%, whereas, the combination test showed 96% specificity and 80 to 93% sensitivity. Cross-reactions were observed commonly between the Dobrava and the Hantaan virus tests, but only rarely between the Puumala and the Hantaan virus, or the Puumala and the Dobrava virus, tests. Altogether, the rapid tests showed less cross-reactivity than the respective EIA tests. According to the results, the performance of these tests meets well the requirements for diagnostic use. Nevertheless, the specific one-antigen tests were markedly more sensitive than the combination test. However, if optimized, a combination test would be suitable for regions where several hantaviruses circulate.     

Hantavirus infections

Over the past few decades understanding and recognition of hantavirus infection has greatly improved worldwide, but both the amplitude and the magnitude of hantavirus outbreaks have been increasing. Several novel hantaviruses with unknown pathogenic potential have been identified in a variety of insectivore hosts. With the new hosts, new geographical distributions of hantaviruses have also been discovered and several new species were found in Africa. Hantavirus infection in humans can result in two clinical syndromes: haemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS) caused by Old World and New World hantaviruses, respectively. The clinical presentation of HFRS varies from subclinical, mild, and moderate to severe, depending in part on the causative agent of the disease. In general, HFRS caused by Hantaan virus, Amur virus and Dobrava virus are more severe with mortality rates from 5 to 15%, whereas Seoul virus causes moderate and Puumala virus and Saaremaa virus cause mild forms of disease with mortality rates <1%. The central phenomena behind the pathogenesis of both HFRS and HCPS are increased vascular permeability and acute thrombocytopenia. The pathogenesis is likely to be a complex multifactorial process that includes contributions from immune responses, platelet dysfunction and the deregulation of endothelial cell barrier functions. Also a genetic predisposition, related to HLA type, seems to be important for the severity of the disease. As there is no effective treatment or vaccine approved for use in the USA and Europe, public awareness and precautionary measures are the only ways to minimize the risk of hantavirus disease.     

汉坦病毒山东分离株的分子生物学研究

目的 应用聚合酶链反应(PCR)、核苷酸序列分析等方法，比较山东省家鼠型汉坦病毒的生物学特性。方法 提取汉坦病毒感染细胞的全细胞RNA，逆转录后用聚合酶链反应扩增病毒cDNA。对PCR产物进行核苷酸序列测定。结果 汉坦病毒山东分离株SD25、SD70、SD27和SDl0均为家鼠型汉坦病毒，核苷酸及氨基酸序列与已知SE0型病毒具有较高的同源性，这4株病毒间的同源性高达98％以上。结论 4株病毒分离时间跨度虽有十几年，但相互间变异很小，证实了家鼠型汉坦病毒的稳定性。     

Development and optimization of a PCR assay for detection of Dobrava and Puumala hantaviruses in Bosnia and Herzegovina

Hantavirus-specific serology tests are the main diagnostic technique for detection of hantavirus infection in Bosnia and Herzegovina. In order to enhance hantavirus infections monitoring a sensitive PCR based assay was developed to detect Dobrava (DOBV) and Puumala (PUUV) hantaviruses. Nested primer sets were designed within three different regions of the viral RNA (S and M segment of DOBV and M segment of PUUV) based on highly similar regions from a number of different European hantavirus strains. Assay conditions were optimized using cell cultures infected with DOBV Slovenia, PUUV Sotkamo and PUUV CG 18-20. This sensitive and specific assay has proven to be useful for detection of both Puumala and Dobrava hantaviruses.     

Hantavirus Infections in Latvia

In order to investigate the presence of hantavirus infections in Latvia, 333 randomly selected human serum samples were screened using an enzyme-linked immunoassay. Fifteen samples were positive for hantavirus-specific IgG and were subsequently serotyped using a focus reduction neutralization test. Fourteen of these samples neutralized at least one of the hantaviruses included in the test, indicating a 4.2% overall seroprevalence in Latvia. Among 14 focus reduction neutralization test-positive sera, specific reactivity (at least 4-fold higher endpoint titer) of neutralizing antibodies was as follows: six sera were specific for Saaremaa hantavirus, three showed equal titers to Saaremaa and Dobrava hantaviruses, and five showed the highest endpoint titers to Puumala hantavirus. Hantavirus infections were confirmed in individuals residing in 11 of 26 investigated counties. The sex ratio was 1:2.5 (M:F), and the antibody prevalence increased with age. This is the first report on the presence of hantavirus infections in Latvia, and the results indicate that two hantaviruses pathogenic to man, Saaremaa virus and Puumala virus, are widely distributed in this country. Electronic Publication     

A new Hantaan-like virus in rodents (Apodemus peninsulae) from Northeastern China

Lung tissue samples of 76 Korean field mice (Apodemus peninsulae) collected from northeastern China bordering with Far East Russia and Korea were detected for hantavirus partial M-segment or entire S-segment sequences by RT-PCR and 481-nt mitochondrial DNA fragment of the rodents. Four A. peninsulae mice were found positive for partial M-segment of hantavirus. Sequence analyses of partial M-segment or/and entire S-segment of the hantaviruses revealed that three were closely related to Hantaan virus (HTNV) strain 76-118. One new variant of HTNV-like virus designated as “Jilin-AP06” was much different from other rodent-borne hantavirus from China, and clustered with Amur (AMR) virus strains, which represent a distinct genetic lineage. These findings imply that hantavirus Jililn-AP06 strain from A. peninsulae is a new record of rodent-borne AMR virus in China. A. peninsulae might be a natural carrier of two distinct hantaviruses, AMR virus and HTNV in China.     

New Ecological Aspects of Hantavirus Infection: A Change of A Paradigm and a Challenge of Prevention- A Review

In the last decades a significant number of so far unknown or underestimated pathogens have emerged as fundamental health hazards of the human population despite intensive research and exceptional efforts of modern medicine to embank and eradicate infectious diseases. Almost all incidents caused by such emerging pathogens could be ascribed to agents that are zoonotic or expanded their host range and crossed species barriers. Many different factors influence the status of a pathogen to remain unnoticed or evolves into a worldwide threat. The ability of an infectious agent to adapt to changing environmental conditions and variations in human behavior, population development, nutrition, education, social, and health status are relevant factors affecting the correlation between pathogen and host. Hantaviruses belong to the emerging pathogens having gained more and more attention in the last decades. These viruses are members of the family Bunyaviridae and are grouped into a separate genus known as Hantavirus. The serotypes Hantaan (HTN), Seoul (SEO), Puumala (PUU), and Dobrava (DOB) virus predominantly cause hemorrhagic fever with renal syndrome (HFRS), a disease characterized by renal failure, hemorrhages, and shock. In the recent past, many hantavirus isolates have been identified and classified in hitherto unaffected geographic regions in the New World (North, Middle, and South America) with characteristic features affecting the lungs of infected individuals and causing an acute pulmonary syndrome. Hantavirus outbreaks in the United States of America at the beginning of the 10th decade of the last century fundamentally changed our knowledge about the appearance of the hantavirus specific clinical picture, mortality, origin, and transmission route in human beings. The hantavirus pulmonary syndrome (HPS) was first recognized in 1993 in the Four Corners Region of the United States and had a lethality of more than 50%. Although the causative virus was first termed in connection with the geographic name of its outbreak region the analysis of the individual viruses indicate that the causing virus of HPS was a genetically distinct hantavirus and consequently termed as Sin Nombre virus. Hantaviruses are distributed worldwide and are assumed to share a long time period of co-evolution with specific rodent species as their natural reservoir. The degree of relatedness between virus serotypes normally coincides with the relatedness between their respective hosts. There are no known diseases that are associated with hantavirus infections in rodents underlining the amicable relationship between virus and host developed by mutual interaction in hundreds of thousands of years. Although rodents are the major reservoir, antibodies against hantaviruses are also present in domestic and wild animals like cats, dogs, pigs, cattle, and deer. Domestic animals and rodents live jointly in a similar habitat. Therefore the transmission of hantaviruses from rodents to domestic animals seems to be possible, if the target organs, tissues, and cell parenchyma of the co-habitat domestic animals possess adequate virus receptors and are suitable for hantavirus entry and replication. The most likely incidental infection of species other than rodents as for example humans turns hantaviruses from harmless to life-threatening pathogenic agents focusing the attention on this virus group, their ecology and evolution in order to prevent the human population from a serious health risk. Much more studies on the influence of non-natural hosts on the ecology of hantaviruses are needed to understand the directions that the hantavirus evolution could pursue. At least, domestic animals that share their environmental habitat with rodents and humans particularly in areas known as high endemic hantavirus regions have to be copiously screened. Each transfer of hantaviruses from their original natural hosts to other often incidental hosts is accompanied by a change of ecology, a change of environment, a modulation of numerous factors probably influencing the pathogenicity and virulence of the virus. The new environment exerts a modified evolutionary pressure on the virus forcing it to adapt and probably to adopt a form that is much more dangerous for other host species compared to the original one.     

Hantavirus infections in Spain: analysis of sera from the general population and from patients with pneumonia, renal disease and hepatitis.

BACKGROUND: Hantaviruses are rodent borne viruses in the family Bunyaviridae that cause significant morbidity in large areas of Europe. There are only a few reports available on hantavirus infections from Spain. Although the results of these earlier studies indicated the presence of hantavirus infections, no confirmative or serotype-specific analyses have been performed. OBJECTIVES: To investigate whether hantaviruses cause human infection/disease in Spain. STUDY DESIGN: Ten thousand, four hundred and eighteen serum samples from the general population and 599 sera from 492 patients with potential hantavirus infections (renal disease, pneumonia or hepatitis) were initially screened by immunofluorescence assay (IFA) using Hantaan, Seoul and Puumala hantavirus antigens. Altogether 193 suspicious samples (165 from healthy people and 28 from patients) were selected for confirmation by quality-assured assays. RESULTS AND CONCLUSIONS: Of the 165 pre-screened serum samples from healthy individuals, only five could be confirmed by IFA for hantavirus-reactive antibodies (using Dobrava, Saaremaa, Hantaan or Puumala virus antigens). In addition, one serum was found weakly positive for hantavirus-reactive IgG by ELISA using recombinant Saaremaa virus (SAAV) nucleocapsid (N) antigen, and subsequently confirmed by immunoblotting. Thus, the results indicated a low (0.06%) total antibody prevalence to hantaviruses in Spain. Of 28 pre-screened serum samples from hospitalized patients, eight reacted as positive or showed border-line reactivities for hantavirus-specific IgM by ELISA using recombinant Saaremaa and Puumala virus N antigens. The IFA/ELISA reactive/border-line samples were subsequently analyzed by a focus reduction neutralization test, which revealed low titers (1:80) against SAAV in two samples from a patient with hepatic disease. The nature of the hantavirus(es) potentially involved remain, however, unknown, since none of the positive samples showed neutralizing titers of the expected range to any of the known European hantaviruses.     

Sequence analysis of the complete S genomic segment of a newly identified hantavirus isolated from the white-footed mouse (Peromyscus leucopus): Phylogenetic relationship with other sigmodontine rodent-borne hantaviruses

Four Corner (FC) or Sin Nombre virus, a hantavirus harbored by the deer mouse (Peromyscus maniculatus), is the principal etiologic agent of hantavirus pulmonary syndrome (HPS). Recently, a hantavirus, designated New York (NY) virus, isolated from a white-footed mouse (Peromyscus leucopus) captured on Shelter Island, New York, was molecularly linked to a fatal case of HPS occurring in the northeastern United States. To clarify the genetic and phylogenetic relationship between NY and FC viruses and other sigmodontine rodent-borne hantaviruses, we amplified and sequenced the entire S genomic segment of NY virus. The S segment of NY virus was 2078 nucleotides long, with an open reading frame of 1284 nucleotides in the virus complementary strand, capable of encoding a protein of 428 amino acids, and with a 752-nucleotide long 3-noncoding region, comprised of numerous imperfect repeats. Pairwise analysis indicated that NY virus was more similar to FC virus than to other sigmodontine rodent-borne hantaviruses, differing from strains of FC virus by 16.6–17.8% and 7.0–8.2% at the nucleotide and amino acid levels, respectively. As determined by the maximum parsimony and neighbor-joining methods, NY virus formed a separate lineage from FC virus and was phylogenetically distinct from hantaviruses harbored by other sigmodontine rodents. Whether or not NY and FC viruses represent distinct viral species is unclear. Further analyses of hantaviruses harbored by white-footed mice are needed to clarify the genetic diversity and evolution of Peromyscus-borne hantaviruses.     

Rodent host specificity of European hantaviruses: evidence of Puumala virus interspecific spillover

In order to investigate rodent host specificity of European hantaviruses, experimental infection of colonized and wild-trapped rodents was performed. In addition to the natural rodent reservoir, Clethrionomys glareolus, Puumala hantavirus (PUUV) could infect colonized Microtus agrestis and Lemmus sibiricus, but not Syrian hamsters or Balb/C mice. Neither C. glareolus, nor M. agrestis, could be readily infected by Tula hantavirus (TULV). Wild-trapped Apodemus flavicollis and A. agrarius, the natural reservoirs of Dobrava (DOBV) and Saaremaa (SAAV) hantaviruses, respectively, could both be infected by SAAV. NMRI mice could also be infected by SAAV, but with lower efficiency as compared to Apodemus mice. Balb/C and NMRI laboratory mice, but not C. glareolus, could be infected by DOBV. To our knowledge, this is the first time DOBV and SAAV have been shown to infect adult laboratory mice. Moreover, potential hantavirus spillover infections were investigated in wild-trapped rodents. In addition to the natural host C. glareolus, we also found M. arvalis and A. sylvaticus with a history of PUUV infection. We did not find any C. glareolus or A. sylvaticus infected with TULV, a hantavirus which is known to circulate in the same geographical regions of Belgium.     

Serological diagnosis of hantavirus infections by an enzyme-linked immunosorbent assay based on detection of immunoglobulin G and M responses to recombinant nucleocapsid proteins of five viral serotypes.

Worldwide, hantaviruses cause more than 100,000 human infections annually. Rapid and accurate methods are important both in monitoring acute infections and for epidemiological studies. We and others have shown that the amino termini of hantavirus nucleocapsid proteins (Ns) are sensitive tools for the detection of specific antibodies in hantavirus disease. Accordingly, we expressed truncated Ns (amino acids 1 to 117) in Escherichia coli from the five hantaviruses known to be pathogenic to man; Hantaan (HTN), Seoul (SEO), Dobrava (DOB), Sin Nombre (SN), and Puumala (PUU) viruses. In order to obtain pure antigens for use in an enzyme-linked immunosorbent assay (ELISA), the recombinant proteins were purified by polyhistidine-metal chelate affinity chromatography. Polyclonal animal antisera and a panel of serum specimens from hantavirus-infected individuals from Scandinavia, Slovenia, Russia, Korea, China, and the United States were used to evaluate the usefulness of the method. With both human and animal sera, it was possible to designate the antibody response into two groups: those with HTN, SEO, and DOB virus reactivity on the one hand and those with SN and PUU virus reactivity on the other. In sera from Scandinavia, European Russia, and the United States, the antibody response was directed mainly to the PUU and SN virus group. The sera from Asia reacted almost exclusively with the HTN, SEO, and DOB types of viruses. This was true for both the immunoglobulin M (IgM) and IgG antibody responses, indicating that this type of discrimination can be done during the acute phase of hantavirus infections. Both the HTN, SEO, and DOB virus and the PUU and SN virus types of antibody response patterns were found in patients from the Balkan region (Solvenia).     

Detection of a new natural virus focus Dobrava in the Astrakhan Region

A natural focus of Dobrava hantavirus was first revealed in an arid zone of the Astrakhan Region, by using molecular genetic methods. A polymerase chain reaction was employed to examine 389 lung tissue suspension samples taken from 9 species. Hantavirus RNA was found in 35 samples from 22 field mice (Apodemus agrarius), 8 tamarisk gerbils (Meriones temariscinus), 4 common voles (Microtus arvalis), and 1 house mouse (Mus musculus). Sequencing determined the taxonomic affiliation of 18 new isolates to the Dobrava species and 1 isolate to the Puumala species although the new Dobrava isolated greatly differed from all known strains of this virus in the nucleotide sequence of the genomic M and S segments. Hantavirus-infected rodents were found in 3 of 4 districts of the Astrakhan Region, located in the steppe and semidesert zones and at the intrazonal stations of the Volga-Akhtuba flood-lands. The high (up to 12%) hantavirus infection rates were ascertained in a tamarisk gerbil, the typical inhabitant of desert and semidesert ecosystems.     

Identification of genetic evidence for dobrava virus spillover in rodents by nested reverse transcription (RT)-PCR and TaqMan RT-PCR

A survey of 158 rodents caught in the Czech Republic identified Dobrava virus sequences closely related to that of the Dobrava virus type strain in Apodemus sylvaticus and Mus musculus rodents. The identity of A. sylvaticus was unequivocally confirmed by random amplified polymorphic DNA analysis. The data seem to indicate hantavirus spillover from Apodemus flavicollis to other rodents.