Puumala virus (PUUV), a causative agent of hemorrhagic fever with renal syndrome (HFRS), is prevalent in Europe and European Russia. No vaccine has been developed for PUUV-associated HFRS, primarily because of the low viral yield in cultured cells. A PUUV strain known as DTK/Ufa-97 was isolated in Russia and adapted for growth in Vero E6 cells maintained in serum-free medium. The DTK/Ufa-97 strain produced a higher viral titer in serum-free medium, suggesting that it may prove useful in the development of an HFRS vaccine. When PUUV-infected Vero E6 cells were grown in serum-free medium, the DTK/Ufa-97 strain yielded more copies of intracellular viral RNA and a higher viral titer in the culture fluid than did the Sotkamo strain. Phylogenetic analysis revealed that PUUVs can be classified into multiple lineages according to geographical origin, and that the DTK/Ufa-97 strain is a member of the Bashkiria-Saratov lineage. The deduced amino acid sequences of the small, medium, and large segments of the DTK/Ufa-97 strain were 99.2% to 100%, 99.3% to 99.8%, and 99.8% identical, respectively, to those of the Bashkirian PUUV strains and 96.9%, 92.6%, and 97.4% identical, respectively, to those of the Sotkamo strain, indicating that the PUUVs are genetically diverse. However, DTK/Ufa-97 and other strains of PUUV exhibited similar patterns of binding to a panel of monoclonal antibodies against Hantaan virus. In addition, diluted antisera (i.e., ranging from 1:160 to 1:640) specific to three strains of PUUV neutralized both homologous and heterologous viruses. These results suggest that the DTK/Ufa-97 strain is capable of extensive growth and is antigenically similar to genetically distant strains of PUUV.

Hantaviruses are causative agents of some severe human illnesses, including hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). The viruses are maintained by rodent hosts, and humans acquire infection by inhaling virus-contaminated excreta from infected animals. To examine the epidemiology of hantavirus infections in Japan and Far East Russia, we conducted epidemiological surveys in these regions. In Japan, anti-hantavirus antibodies were found in four rodent species, Clethrionomys rufocanus, Rattus norvegicus, R. rattus, and Apodemus speciosus. Although no new HFRS cases have been officially reported over the past 20 years in Japan, one member of the Japan Ground Self-Defense Force did test positive for hantavirus antibody. Repeated surveys in Far East Russia have revealed that two distinct hantavirus types cause severe HFRS in this region. Hantavirus sequences identified from A. peninsulae, fetal HFRS cases in Vladivostok, and Amur virus are highly similar to each other (92% identity) , but they are less similar (-84% identity) to the prototypical Hantaan virus, which is carried by A. agrarius. Phylogenetic analysis also indicates that Amur and A. peninsulae -associated viruses are distinct from Hantaan virus, suggesting that A. peninsulae is the reservoir animal for Amur virus, which causes severe HFRS. From HFRS patients in the Khabarovsk region, we identified viruses with nucleotide sequences that are more similar to Far East virus (96%identity) than to the Hantaan (88-89%identity) or Amur (81-83% identity) viruses. Phylogenetic analysis also indicates that the viruses from Khabarovsk HFRS patients are closely related to the Far East virus, and distinct from Amur virus.

Hantaviral antibodies were detected in the sera from Apodemus (A.) agrarius and A. peninsulae captured in Ningxia province, China by several different serological diagnostic methods. A total of 409 sera from rodent and insectivore species were collected in 1999 and examined by indirect immunofluorescent antibody assay (IFA). Among them, 19 of 191 (9.9%) sera of A. agrarius and 1 of 13 (7.7%) sera of A. peninsulae were positive for hantaviral antibodies. The other species (Rattus norvegicus, Mus musculus, Cricetulus triton, and Sorex cylindricauda) were negative. The reaction pattern of positive serum was characterized as scattered and granular virus antigens in the cytoplasm of hantavirus infected Vero E6 cells. Some of the A. agrarius sera positive for hantavirus were further examined by Western blotting (WB), enzyme-linked immunosorbent assay (ELISA), and the focus reduction neutralization test FRNT By WB, positive sera showed the same specific reaction pattern of baculovirus-expressed recombinant hantaviral nucleocapsid protein, as shown in hantavirus-immune serum. By ELISA, IFA-positive sera showed significantly higher optical densities (around 1.0) than the negative A. agrarius sera. Hantaan type hantavirus was neutralized with the positive sera. These results suggest that A. agrarius have hantavirus infection and may play a role as a reservoir animal for hantavirus in Ningxia Hui Autonomous Province, China.