Angelina Plyusnina

Isolation and characterization of Dobrava hantavirus carried by the striped field mouse (Apodemus agrarius) in Estonia

Dobrava hantavirus (DOB) was isolated from the striped field mouse (Apodemus agrarius) trapped on Saaremaa Island, Estonia, and its genetic and antigenic characteristics were subsequently analysed. Phylogenetic analysis showed that the Estonian DOB strain, together with several wild strains carried by Apodemus agrarius, forms a well-supported lineage within the DOB clade. The topography of the trees calculated for the S, M and L nucleotide sequences of the Estonian DOB suggests a similar evolutionary history for all three genes of this virus and, therefore, the absence of heterologous reassortment in its evolution. A cross-neutralization comparison of the Estonian virus with the prototype DOB, isolated from a yellow-necked mouse (A. flavicollis) in Slovenia, revealed 2- to 4-fold differences in the end-point titres of rabbit and human antisera. When studied with a panel of 25 monoclonal antibodies (MAbs), the Estonian and Slovenian DOB isolates showed similar antigenic patterns that could be distinguished by two MAbs. Genetic comparison showed sequence differences in all three genome segments of the two DOB isolates, including an additional N-glycosylation site in the deduced sequence of the G2 protein from the Estonian virus. Whether any of these mutations relates to the different rodent hosts rather than to the distant geographical origin of the two isolates remains to be resolved. Taken together, our observations suggest that A. agrarius, which is known to harbour Hantaan virus in Asia, carries another hantavirus, DOB, in north-east Europe.

Transfection-mediated generation of functionally competent Tula hantavirus with recombinant S RNA segment

Since the discovery of RNA recombination in polioviruses, there has been a general belief that this mechanism operates only in positive‐sense RNA viruses. Recently, studying wild‐type Tula hantavirus, we observed a mosaic‐like structure of the S RNA segment that was consistent with generation by recombination between viruses from two genetic lineages. Here we show transfection‐mediated rescue of Tula virus carrying recombinant S RNA segment. Independent attempts yielded S RNA molecules of similar structure; the majority of them carried a break point located close to one of the break points suggested for natural recombinants. Recombinant virus purified from the original variant was able to grow to the same titers in cell culture and showed the same characteristic immunofluorescence pattern when stained for the nucleocapsid protein. While competent, the recombinant virus appeared to be slightly less competitive than the wild type. Sequence analysis of the S cDNA clones obtained from the purified recombinant virus confirmed that all S RNA molecules were of recombinant origin. This provides the first example of a negative‐sense RNA virus constructed using homologous recombination.

Analysis of Puumala hantavirus in a bank vole population in northern Finland: evidence for co-circulation of two genetic lineages and frequent reassortment between strains

In this study, for the first time, two distinct genetic lineages of Puumala virus (PUUV) were found within a small sampling area and within a single host genetic lineage (Ural mtDNA) at Pallasjärvi, northern Finland. Lung tissue samples of 171 bank voles (Myodes glareolus) trapped in September 1998 were screened for the presence of PUUV nucleocapsid antigen and 25 were found to be positive. Partial sequences of the PUUV small (S), medium (M) and large (L) genome segments were recovered from these samples using RT-PCR. Phylogenetic analysis revealed two genetic groups of PUUV sequences that belonged to the Finnish and north Scandinavian lineages. This presented a unique opportunity to study inter-lineage reassortment in PUUV; indeed, 32 % of the studied bank voles appeared to carry reassortant virus genomes. Thus, the frequency of inter-lineage reassortment in PUUV was comparable to that of intra-lineage reassortment observed previously (Razzauti, M., Plyusnina, A., Henttonen, H. & Plyusnin, A. (2008). J Gen Virol 89, 1649-1660). Of six possible reassortant S/M/L combinations, only two were found at Pallasjärvi and, notably, in all reassortants, both S and L segments originated from the same genetic lineage, suggesting a non-random pattern for the reassortment. These findings are discussed in connection to PUUV evolution in Fennoscandia.

Isolation and characterization of Puumala hantavirus from Norway: evidence for a distinct phylogenetic sublineage

Puumala (PUU) hantavirus is the aetiological agent of nephropathia epidemica (NE), a mild form of haemorrhagic fever with renal syndrome, which occurs in Fennoscandia, central Europe and Russia. In Norway, NE-like disease has been reported since 1946 and about 50 cases are diagnosed annually; however, the causative agent has not been characterized. In this study, a virus originating from bank voles (Clethrionomys glareolus) trapped near the town of Eidsvoll (Akershus county) was isolated and passaged in laboratory-bred bank voles. The bank vole strain was identified as a PUU virus by serological typing and by sequence analysis of the S and M gene segments. For comparison, complete or partial S sequences were determined for wild-type PUU strains from five locations in Sweden, two inhabited by the southern variant of bank vole present in Fennoscandia, and three by the northern variant. Phylogenetic analysis showed that Norwegian PUU strains are clustered together with Swedish strains from the first group forming a well-supported sublineage within the PUU genotype, distinct from other sublineages from northern Sweden, Finland, Russia and France. The results are consistent with the view of a complex evolutionary history of PUU strains in post-glacial Fennoscandia. Analyses of the current collection of nucleotide sequences suggest that PUU is the most variable genotype of the known hantaviruses.

Identification of Seoul hantavirus in Rattus norvegicus in Indonesia.

The first genetic evidence for the presence of Seoul hantavirus (SEOV) in Indonesia is presented. Partial M segment sequence was recovered from the lung tissue of Rattus norvegicus trapped in central Jakarta. The sequence belongs to SEOV genotype and is most closely related to the strain B-1 from Japan.

Tula hantavirus isolate with the full-length ORF for nonstructural protein NSs survives for more consequent passages in interferon-competent cells than the isolate having truncated NSs ORF

BackgroundThe competitiveness of two Tula hantavirus (TULV) isolates, TULV/Lodz and TULV/Moravia, was evaluated in interferon (IFN) -competent and IFN-deficient cells. The two isolates differ in the length of the open reading frame (ORF) encoding the nonstructural protein NSs, which has previously been shown to inhibit IFN response in infected cells.ResultsIn IFN-deficient Vero E6 cells both TULV isolates survived equally well. In contrast, in IFN-competent MRC5 cells TULV/Lodz isolate, that possesses the NSs ORF for the full-length protein of 90 aa, survived for more consequent passages than TULV/Moravia isolate, which contains the ORF for truncated NSs protein (66–67 aa).ConclusionOur data show that expression of a full-length NSs protein is beneficial for the virus survival and competitiveness in IFN-competent cells and not essential in IFN-deficient cells. These results suggest that the N-terminal aa residues are important for the full activity of the NSs protein.

Serological and genetic evidence for the presence of Seoul hantavirus in Rattus norvegicus in Flanders, Belgium

Seoul hantavirus (SEOV), carried by Rattus rattus (black rat) and R. norvegicus (Norway, brown rat), was reported to circulate as well as cause HFRS cases in Asia. As Rattus sp. are present worldwide, SEOV has the potential to cause human disease worldwide. In Europe however, only SEOV prevalence in rats from France was reported and no confirmed cases of SEOV infection were published. We here report genetic and serological evidence for the presence of SEOV virus in brown rat populations in Belgium. We also serologically screened an at-risk group that was in contact with R. norvegicus on a daily basis and found no evidence for SEOV infection.

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.

Genetic analysis of hantaviruses carried by Myodes and Microtus rodents in Buryatia

Hantavirus genome sequences were recovered from tissue samples of Myodes rufocanus, Microtus fortis and Microtus oeconomus captured in the Baikal area of Buryatia, Russian Federation. Genetic analysis of S- and M-segment sequences of Buryatian hantavirus strains showed that Myodes- associated strains belong to Hokkaido virus (HOKV) type while Microtus- associated strains belong to Vladivostok virus (VLAV) type. On phylogenetic trees Buryatian HOKV strains were clustered together with M. rufocanus- originated strains from Japan, China and Far-East Russia (Primorsky region). Buryatian Microtus- originated strains shared a common recent ancestor with M. fortis- originated VLAV strain from Far-East Russia (Vladivostok area). Our data (i) confirm that M. rufocanus carries a hantavirus which is similar to but distinct from both Puumala virus carried by M. glareolus and Muju virus associated with M. regulus, (ii) confirm that M. fortis is the natural host for VLAV, and (iii) suggest M. oeconomus as an alternative host for VLAV.

Genetic analysis of Thailand hantavirus in Bandicota indica trapped in Thailand

Sixty one tissue samples from several rodent species trapped in five provinces of Thailand were examined for the presence of hantaviral markers by enzyme-immunoassay and immunoblotting. Four samples, all from the great bandicoot rat Bandicota indica, were confirmed positive for the hantaviral N-antigen. Two of them were trapped in Nakhon Pathom province, the other two in Nakhon Ratchasima province, approximately 250 km from the other trapping site. When analysed by RT-nested PCR, all four rodents were found positive for the hantaviral S- and M-segment nucleotide sequences. Genetic analysis revealed that the four newly described wild-type strains belong to Thailand hantavirus. On the phylogenetic trees they formed a well-supported cluster within the group of Murinae-associated hantaviruses and shared a recent common ancestor with Seoul virus.