Comparison of virulence of various hantaviruses related to hemorrhagic fever with renal syndrome in newborn mouse model

The virulence of hantaviruses that are antigenically related but have different genetic characteristics from the prototype of hantavirus, Hantaan (HTN) virus, was examined in newborn mice. The H5 and B78 strains of the Amur (AMR) genotype, the Bao14 strain of the Far East (FE) genotype, and the 76-118 strain of HTN virus were inoculated subcutaneously (1focus-forming unit; FFU) into newborn mice. All of the AMR and FE genotype viruses inoculated mice were died by 16 days post-infection (dpi) and 21 dpi, respectively, while 50% of the HTN virus inoculated mice survived until 30 dpi. The AMR and FE genotype viruses inoculated mice had high viral titers in the lung (1.3x10(6) to 1.3x10(8) FFU/gram [g] tissue) , brain (2.1x10(7) to 1.2x10(9) FFU/g tissue), and kidney(2.5x10(5) to 1.6x10(7) FFU/g tissue), and showed a detectable level of antibodies (titers 1:16-1:32) at 14 dpi. In contrast, the HTN virus infected mice had viruses only in the lungs at low titers (1.1-5.3x10(5) FFU/g tissue). Observations of body-weight changes revealed that the AMR and FE genotype viruses inoculated mice had lower growth rates than the HTN virus inoculated mice. These data suggest that the AMR and FE genotype viruses are more virulent than the HTN virus in newborn mice.     

Development of an efficient method for recovery of Puumala and Puumala-related viruses by inoculation of Mongolian gerbils

Puumala (PUU) virus and PUU-related viruses are difficult to isolate in cell culture. To determine whether animal inoculation would be a better alternative for virus recovery, the Sotkamo strain of PUU virus was inoculated into several animal species. Newborn Mongolian gerbils (MGs), mice, and rats were infected with the Sotkamo strain by intracerebral (ic), intraperitoneal (ip), and subcutaneous (sc) inoculation. Antibodies to PUU appeared in MGs at 30 days post-infection (dpi), and in mice and rats at 15 dpi. Interestingly, virus appeared at 7 dpi in lung and brain of MGs inoculated via ic and ip routes. Virus was detected in all tested tissues of MGs at 15 dpi, with a peak level of 1.36 x 10 (5) focus forming units (FFU)/g in brain tissue. The virus titer declined with the onset of the antibody response and became undetectable by 75 dpi, when the antibody titer reached the maximum level. The appearance of the virus in mice and rats was delayed as compared to MGs, and the virus titer was apparently lower, at approximately 4 to 8 x 10(3) FFU/g, at 15 dpi. In addition, lung homogenates of antibody-positive Clethrionomys (C.) rufocanus (captured in Tobetsu, Hokkaido, Japan) were inoculated into MGs by the ic route. PUU-related viral RNA was detected at 16 dpi in the brains of MG inoculated with the lung homogenate, and antibodies were detected at 45 dpi. These findings indicate that newborn MG inoculation is an efficient method to recover PUU and PUU-related viruses.     

A comparative epidemiological study of hantavirus infection in Japan and Far East Russia

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 (approximately 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.     

Epizootiological survey of hantavirus among rodent species in Ningxia Hui Autonomous Province, China

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.