Modes of hantavirus transmission in a population of Clethrionomys rufocanus bedfordiae inferred from mitochondrial and microsatellite DNA analyses.

To elucidate the mode of transmission of Puumala-related hantavirus in a population of gray red-backed voles, Clethrionomys rufocanus bedfordiae, in Hokkaido, Japan, we analyzed the kin structure and dispersal patterns of individual voles using microsatellite and mitochondrial DNA markers. Siblings or dam/offsprings was identified within the population based on the relatedness calculation with the microsatellite data. The pairwise relatedness values obtained could reveal kinship among all vole individuals within the population. Based on the assessment of kinship, we did not find a positive relationship between hantavirus transmission and close kinship. Males infected with the hantavirus carried a relatively uncommon mitochondrial haplotype. However, these infected males shared low relatedness values and were not considered closely related, i.e., they were not siblings or parent/offspring. These observations imply that hantavirus transmission in the vole population may not be related to close kinship but by random horizontal infection.

Synthesis of Seoul virus RNA and structural proteins in cultured cells.

Seoul virus is a hantavirus that causes hemorrhagic fever with renal syndrome (HFRS). The virion has a tripartite (S, M, and L) negative-stranded RNA genome, which is characteristic of the family Bunyaviridae. However, the molecular basis of virus replication is not well known. We established a Northern blot hybridization (NB) procedure using digoxygenin-labeled RNA probes, to quantitate the hantaviral plus- and minus-strand RNAs separately. Virus RNA replication was analyzed in infected Vero E6 cells. When the Vero E6 cells were infected with Seoul virus strain KI-83-262 (KI) at m.o.i. = 0.25, the plus-strand RNA was detected within 1 h post-infection (hpi), and the minus-strand RNA was detected subsequently. Using laser confocal microscopy, the nucleocapsid protein (NP) was detected within 2 hpi, and accumulated as scattered granules in the cytoplasm until 24 hpi. In contrast, the G2 protein first appeared at 8 hpi, was immediately transported to the Golgi, and accumulated in the Golgi until 24 hpi. Infectious virus particles were released into the medium at 24 h hpi. These findings indicate that hantavirus RNA replication starts with the appearance of NP at 2 hpi, glycoproteins then accumulate gradually in the Golgi, and virion formation is initiated once the viral RNAs and proteins have accumulated.

Serological analysis of hemorrhagic fever with renal syndrome (HFRS) patients in Far Eastern Russia and identification of the causative hantavirus genotype.

Hemorrhagic fever with renal syndrome (HFRS) is endemic in East Asia and Europe. The disease is caused by several viruses belonging to the genus Hantavirus, including the Hantaan virus (HTNV), Seoul virus (SEOV), Dobrava Belgrade virus (DOBV), and Puumala virus (PUUV). Recently, HTNV-related viruses, Amur (AMR) and Far East (FE) genotypes were identified as causative agents of HFRS in Far Eastern Russia. To investigate the epidemiology of HFRS and virus transmission, we collected sera from 17 acute and 32 convalescent patients who were clinically diagnosed with HFRS in the Khabarovsk region of Far Eastern Russia, and detected anti-hantavirus antibodies using an ELISA that can differentiate the infected virus serotype using truncated hantavirus nucleocapsid protein antigen. Sixteen of the 17 acute phase patients had antibodies to hantavirus, and all the positive sera had higher optical densities for HTNV-specific antigen than for SEOV-, DOBV-, or PUUV-specific antigens. The partial M segment of the viral genome was amplified from blood clots from three acute patients by PCR. The nucleotide sequences had closer identities to the FE genotype (>96%) than to the prototype HTNV (88 to 89%) or AMR genotype (81 to 83%). A phylogenetic analysis found that the virus sequences from the patients clustered with the FE type, and were distinct from the AMR type. Thirty-one of 32 convalescent patient sera had antibodies to HTNV-specific antigen. These data suggest that our ELISA system can detect HTNV-specific antibodies to the FE type, which may be responsible for most of the HFRS in Khabarovsk.

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.