Molecular characterization and expression of glycoprotein gene of Hantavirus R22 strain isolated from Rattus norvegicus in China.

A cDNA containing the complete open reading frame of the M genome segment of Hantavirus R22 strain isolated from Rattus norvegicus in China, was amplified by polymerase chain reaction (PCR), and then cloned. The M segment is 3656 nucleotides in length with a predicted region of 3402 bases encoding a precursor glycoprotein of 1134 amino acids subsequently processed into viral glycoproteins 1 and 2 (G1 and G2). A strain comparison between R22 and SR11 (isolated from a rat in Japan), and Hantaan 76-118 (isolated from Apodemus in Korea), and Hallnas B1 (isolated from a bank vole in Sweden) revealed 95%, 74%, and 53% homologies at the deduced amino acid sequence level respectively. This suggests that the rodent host species may be a more important determinant of genetic relationships than geographic proximity. Six potential asparagine linked glycosylation sites (five in G1 and one in G2) were identified, and among them all are conserved in SR11, five in Hantaan virus and four in Hallnas B1 virus. Although different degrees of homology exist among these four viruses at amino acid sequence level, more than 90% of the cysteine residues are conserved, suggesting that structural homology may be very strong between the Hantaviruses. Genetic differences in the M segment genome of R22 and SR11 viruses, within the same serotype viruses, were found as random coding changes; some limited to single amino acids, others in clusters. A recombinant vaccinia virus that contained the fully activated M segment cDNA of R22 was constructed. This recombinant virus expressed two glycoproteins G1 and G2 identical to R22 virus G1 and G2 in molecular weight, cleavage pattern and cellular immunofluorescent patterns.

Immunity to Hantavirus challenge in Meriones unguiculatus induced by vaccinia-vectored viral proteins.

Vaccinia virus recombinants were constructed that incorporated genomic sequences coding for the nucleoprotein (N) and glycoproteins (G1 and G2) of the hantavirus R22 strain isolated from a rat in China, and designated as RNV and RMV9, respectively. The proteins expressed by RNV and RMV9 were identified by radioimmunoprecipitation and indirect immunofluorescence assay using a panel of monoclonal antibodies and polyclonal immune sera, and were found to be antigenically indistinguishable from authentic R22 viral proteins. Both RNV and RMV9 elicited an anti-R22 antibody response in Mongolian gerbils (Meriones unguiculatus) with titers ranging from 6,400 to 12,800 by enzyme-linked immunosorbent assay, but only RMV9 produced neutralizing antibodies to R22 virus (titer 1:200) and Hantaan (HTN) virus (titer 1:20). The ability of these recombinants to protect Mongolian gerbils against challenge with R22 and HTN viruses was examined. The RMV9 recombinant induced a complete protective immune response against challenge with 10(4) plaque-forming units (PFU) of both R22 and HTN viruses, while RNV induced partial protection against a challenge with the homologous R22 virus and the heterologous HTN virus at a dose of 10(3) PFU. Our data show that the common antigenic sites responsible for eliciting a protective response are located mainly on hantavirus glycoproteins, and that the nucleoprotein may also confer partial cross-protection that presumably involves cell-mediated as well as humoral mechanisms.

A serotypic study of hemorrhagic fever with renal syndrome in rural China.

Apodemus agrarius was trapped in the fields and Rattus norvegicus was trapped within the houses in the villages of Jiande County, a region in the Zhejiang Province of China endemic for hemorrhagic fever with renal syndrome (HFRS). Antibodies to hantaviruses were detected in three (16.7%) of 18 A. agrarius and 12 (13.5%) of 89 R. norvegicus, whereas hantavirus antigens were detected in the lung tissues of four (22.2%) of 18 and nine (10.1%) of 89 of these rodents, respectively. Three hantaviruses, one from A. agrarius and two from R. norvegicus, were isolated and found to be antigenically similar to Hantaan and Seoul serotype viruses, respectively. A serologic study of 437 clinically defined HFRS patients conducted in Jiande County in 1988 revealed that the ratio of Hantaan (72.5%) to Seoul (26.8%) serotype virus infections was 2.7:1. Two epidemic seasons were found, with a major peak in November and a minor peak in June, and both were associated with Hantaan serotype virus infections that coincided with two seasonal peaks of the A. agrarius population and local agricultural activities in the fields. Seoul serotype virus infections occurred with a small peak during the months of December through May, in which in-house activities were dominant. All data suggested that Jiande County was an area endemic for HFRS, predominantly of the Hantaan virus serotype, combined with Seoul serotype virus infections.

Monoclonal antibodies to three strains of hantaviruses: Hantaan, R22, and Puumala.

Thirty hybrid cell lines that produce monoclonal antibodies to three strains of hantaviruses have been generated and characterized. One clone specific to Hantaan 76-118 strain, four clones specific to Rattus strains and one clone specific to Puumala virus have been identified. Most of the monoclones produced antibodies specific to nucleoproteins. Only two monoclones were found to produce glycoprotein specific, neutralizing antibodies. The immunofluorescent (IFA) staining patterns of the monoclonal antibodies show consistent correlation with viral protein specificities as described for other hemorrhagic fever viruses. Cross-reactivity studies with hantaviruses tested demonstrate conserved antigenic sites on nucleoproteins among these hantaviruses tested. Puumala specific monoclones, produced for the first time, reveal both conserved and strain specific sites on the viral nucleoproteins of the Scandinavian virus.

Antigenic relatedness between arenaviruses defined at the epitope level by monoclonal antibodies.

Monoclonal antibodies (MAbs) were produced against two African arenaviruses, Lassa virus and Mopeia virus. Competitive binding analysis of MAbs identified four antigenic sites on the nucleoprotein (NP), two on glycoprotein 1 (GP1) and six on glycoprotein 2 (GP2) of the Josiah strain of Lassa virus. 64 virus isolates from western, central and southern Africa were all consistently distinguishable by MAbs to certain epitopic sites on GP1, GP2 and NP viral proteins. Furthermore, MAbs to Lassa virus GP1 and NP uniformly distinguished viruses from the West African countries of Sierra Leone, Liberia and Guinea from those of Nigeria. GP2-directed MAbs to two African arenaviruses reacted broadly with South American arenaviruses demonstrating that an epitopic site on GP2 may be the most highly conserved antigen in the arenavirus group.

Distribution of hantavirus serotypes Hantaan and Seoul causing hemorrhagic fever with renal syndrome and identification by hemagglutination inhibition assay.

An epidemiologic evaluation of patients with hemorrhagic fever with renal syndrome from different locations in the People's Republic of China was conducted to define the prevalence of two Hantavirus serotypes, Seoul (SEO) and Hantaan (HTN). Serum specimens were collected between 5 and 14 days after the onset of illness and were tested for antibodies by both hemagglutination inhibition (HI) and plaque reduction neutralization (PRN). By the HI test, the geometric mean titer (GMT) of antibodies to SEO in the sera from individuals from Kaifeng City of Henan Province was five times higher than that to HTN. In contrast, by the HI test, the sera from individuals from Jiande County of Zhejiang Province had a GMT of antibodies to HTN that was seven times higher than that to SEO. In the sera from individuals from Shanghai, only a twofold difference was observed in HI antibody titers to the two hemagglutinins by the HI test, with that to HTN being higher than that to SEO. By the PRN test, the GMT ratios of antibody between HTN and SEO strains from individuals in Kaifeng, Jiande, and Shanghai were found to be 1:13, 14:1, and 2:1 respectively. A close correlation (r = 0.8219) and concordance rate (78.3%) were observed between the PRN and HI tests for the identification of the serotypes of individual cases of hemorrhagic fever with renal syndrome. The hantavirus serotypes from individuals in Kaifeng and Jiande were identified as predominantly SEO and HTN, respectively, and those from individuals in Shanghai had an indeterminant serotype defined by these two techniques. The HI test appears to be a simple and reliable way of determining the predominant hantavirus that causes HFRS in a given geographic area.

Genetic identification of a new Puumala virus strain causing severe hemorrhagic fever with renal syndrome in Germany.

A severe case of suspected hemorrhagic fever with renal syndrome (HFRS) was recently identified in northwestern Germany. A genetic detection assay was designed that identified hantavirus-specific RNA in the patient's clinical specimens by reverse transcriptase-polymerase chain reaction amplification of virus S and M genome segments. Phylogenetic analysis of the nucleotide sequences demonstrated that this virus belonged to the Puumala (PUU) group, with the closest relationship to a PUU isolate from Finland. Within the group, this virus formed a separate lineage. This finding represents the first genetic characterization of a hantavirus causing severe HFRS in Germany. The data suggest that PUU viruses circulating in western European countries are genetically distinct from their northeastern counterparts. Comparison of deduced amino acid sequences demonstrated a loss of a potential N-glycosylation site in the G2 protein compared with other PUU viruses.

Hantavirus strains isolated from rodentia and insectivora in rural China differentiated by polymerase chain reaction assay.

Polymerase chain reaction (PCR) assay and other techniques were applied to differentiate Hantavirus strains isolated from different animal hosts and geographic regions in China. Two groups of related strains, Hantaan and Seoul, have been classified by cross-neutralization, radioimmunoprecipitation (RIP), and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) assays. The molecular weights of glycoprotein 1 (G1) of Hantaan and Seoul viruses were 72k and 80k, whereas those of the nucleocapsid (N) and glycoprotein 2 (G2) remained the same, respectively. The PCR assay was used to differentiate these isolates using synthetic oligonucleotide primers selected from various regions of the M genome of 76118 and R22 strains. 76118-specific primers amplified only the RNAs extracted from Hantaan strains while R22-specific primers, the RNAs from Seoul strains. The PCR results for classification are consistent with those obtained by cross-neutralization, RIP and SDS-PAGE assays.

Pathogenic potential of filoviruses: role of geographic origin of primate host and virus strain.

African filoviruses have caused outbreaks of fulminating hemorrhagic fever among humans. In 1989, related filoviruses were isolated from cynomolgus monkeys imported into the United States from the Philippines. The pathogenic potential of these new filoviruses was compared in 16 Asian monkeys (Macaca fascicularis-cynomolgus) and 16 African monkeys (Cercopithecus aethiops-African green) using African filoviruses from Zaire (Ebola virus) and Sudan or Asian filoviruses (Reston and Pennsylvania). African filovirus infections resulted in earlier death (P = .005), had a shorter duration of disease and median incubation period (3-4 vs. 7 days), and had earlier peak viremia (5-7 vs. 7-9 days). African green monkeys showed significantly higher survival than cynomolgus monkeys (P less than .01), and some were asymptomatic as have been humans accidentally infected with Asian filovirus. Rechallenge experiments showed that protection in survivors of filovirus infections against fatal challenge with Ebola (Zaire) virus is unpredictable. The minimal clinical disease observed in humans infected with the Reston strain is consistent with host- and virus-dependent pathogenicity.

Retrospective and prospective studies of hemorrhagic fever with renal syndrome in rural China.

Residents of two villages in Zhejiang Province, China, were interviewed and serum samples were collected to assess prevalence of hantavirus infection. Antibody prevalence was 12% (219/1811), with a ratio of illness to infection of 1.0:5.4. Seroprevalence increased with age, but no association was found with sex. There was also no evidence of vertical transmission. One year later, 2.3% (30/1325) of seronegative subjects had seroconverted including 2 who had hemorrhagic fever with renal syndrome. Peak incidence of infection occurred in those 15-39 years old. Hantaan was the dominant serotype; Seoul serotype was less common (5:1). Host reservoirs were Apodemus agrarius in agricultural fields and Rattus norvegicus in houses. Risk factors for infection were traces of rat-contaminated food, travel to other areas for farm work, direct rodent contact, camping in grain fields, living in a house on the periphery of a village, stacking straw stacks outside houses, and keeping cats. All may provide exposure to infectious rodent reservoirs.

Hantavirus infection in Taiwan: the experience of a geographically unique area.

Hantaviruses are rodent-borne viruses, and they, mainly the Hantaan (HTN) serotype, are the causative agents of a group of febrile nephropathies known as "hemorrhagic fever with renal syndrome (HFRS). " Despite the fact that HFRS is frequently reported in China, with an annual incidence of 50,000-100,000 cases, one puzzling observation that no local case of HFRS has been confirmed in Taiwan has yet to be explained. We hypothesized that the hantavirus strain prevailing in Taiwan mainly belongs to the mild strain, the Seoul (SEO) strain, and the absence of severe disease was related to the absence of HTN. To test these hypotheses, this epidemiologic study was performed, including a seroprevalence survey and phylogenetic analysis on hantavirus isolated from the rodent population trapped in major seaports, rural, and mountainous areas of Taiwan. This study also included rodents and viruses from two isolated islands, Kinmen and Matzu, which are geographically adjacent to the east coast of mainland China. There were a total of 5,461 rodents of 16 species captured, and R. norvegicus was the most common species, with an antibody prevalence much higher in international seaports (20%) than in rural regions (approximately 5%) and intermediate in some domestic seaports. By reverse transcriptase polymerase chain reaction (RT-PCR), 33.9% of the seropositive R. norvegicus were found to have amplifiable hantavirus sequences in their lung tissues, and subsequent phylogenetic analyses indicated that almost all hantavirus in Taiwan was most closely related to the prototype SEO strain, and no HTN strain was recovered from any rodent species indigenous to Taiwan. The seroprevalence of SEO infection in R. norvegicus on Kinmen and Matzu was also different from that in southern provinces of China but closely resembled that in seaports in Taiwan, and the SEO identified was genetically linked to Taiwanese SEO strains. These results substantiate our hypotheses, and suggest that the epidemiology of hantavirus infection in Taiwan are different from that in China, where the HTN and SEO strains and HFRS concurrently prevail.