Jiro Arikawa

Characterization of Hantaan virus envelope glycoprotein antigenic determinants defined by monoclonal antibodies.

A panel of 24 monoclonal antibodies (MAbs) to the G1 or G2 envelope glycoproteins of Hantaan virus were used to determine the surface topography and functional properties of antigenic sites. Nine distinct, partially overlapping antigenic sites, two on G1 and seven on G2, were demonstrated by competitive binding assays. Analyses of the antigenic sites by haemagglutination (HA) inhibition and plaque-reduction neutralization tests showed that all of the sites, except one on G1, were related to viral HA. Only one of the G1 antigenic sites and two of the G2 sites were involved in virus neutralization. These results suggest that certain epitopes related to HA were not critical for virus neutralization. The nine antigenic sites could be further divided into 13 based upon the serological cross-reactivity of MAbs with viruses representative of each of the four known antigenic groups within the Hantavirus genus of Bunyaviridae, i.e. Hantaan, Seoul, Puumala and Prospect Hill viruses.

Coding properties of the S and the M genome segments of Sapporo rat virus: comparison to other causative agents of hemorrhagic fever with renal syndrome.

Three serologically distinct groups of hantaviruses have been associated with severe, moderate, and mild forms of hemorrhagic fever with renal syndrome (HFRS). To gain a better understanding of the genetic variation among these viruses, we cloned and sequenced the M and the S genome segments of Sapporo rat virus, an etiologic agent of moderate HFRS, and compared the predicted gene products to those of Hantaan virus, and the Hällnäs strain of Puumala virus, which are etiologic agents of severe and mild HFRS, respectively. The SR-11 S segment consisted of 1769 nucleotides and had an open reading frame (ORF) in the virus-complementary sense RNA with a coding capacity of 429 amino acids. Deduced amino acids from the SR-11 S segment ORF displayed 83% homology with those of Hantaan nucleocapsid (N) protein. Comparison of the S segment ORFs of all three viruses revealed 58% homology. No evidence for additional nonstructural protein(s) encoded by the SR-11 S segment was obtained. The SR-11 M segment consisted of 3651 nucleotides and had an ORF in the virus-complementary sense RNA with a coding capacity of 1134 amino acids. Amino acid sequences predicted from the SR-11 M segment ORF were 75% homologous with those encoding Hantaan G1 and G2 envelope glycoproteins. Comparison of the deduced amino acid sequences of the M segment ORFs of SR-11, Hantaan, and Hällnäs viruses revealed a 43% homology for amino acids constituting the G1 proteins and a 55% homology for amino acids constituting the G2 proteins of the three viruses. The envelope proteins of SR-11 virus were localized within the M segment ORF by amino-terminal sequence analysis of purified G1 and G2. G1 initiated at amino acid 17 and G2 at amino acid 647 within the ORF. Five potential asparagine-linked glycosylation sites were identified in the SR-11 G1 coding sequences, four of which were conserved between Hantaan and SR-11 viruses and three of which were conserved among all three viruses. One potential glycosylation site was identified in the SR-11 G2 coding sequences and was conserved among Hantaan, SR-11 and Hällnäs viruses. Cysteine residues were highly conserved within the M segment ORFs of all three viruses, suggesting a similar structure and function of the G1 and G2 proteins.

Molecular phylogeny of a newfound hantavirus in the Japanese shrew mole (Urotrichus talpoides)

Recent molecular evidence of genetically distinct hantaviruses in shrews, captured in widely separated geographical regions, corroborates decades-old reports of hantavirus antigens in shrew tissues. Apart from challenging the conventional view that rodents are the principal reservoir hosts, the recently identified soricid-borne hantaviruses raise the possibility that other soricomorphs, notably talpids, similarly harbor hantaviruses. In analyzing RNA extracts from lung tissues of the Japanese shrew mole (Urotrichus talpoides), captured in Japan between February and April 2008, a hantavirus genome, designated Asama virus (ASAV), was detected by RT-PCR. Pairwise alignment and comparison of the S-, M-, and L-segment nucleotide and amino acid sequences indicated that ASAV was genetically more similar to hantaviruses harbored by shrews than by rodents. However, the predicted secondary structure of the ASAV nucleocapsid protein was similar to that of rodent- and shrew-borne hantaviruses, exhibiting the same coiled-coil helix at the amino terminus. Phylogenetic analyses, using the maximum-likelihood method and other algorithms, consistently placed ASAV with recently identified soricine shrew-borne hantaviruses, suggesting a possible host-switching event in the distant past. The discovery of a mole-borne hantavirus enlarges our concepts about the complex evolutionary history of hantaviruses.

Protective role of antigenic sites on the envelope protein of Hantaan virus defined by monoclonal antibodies

SummaryTo investigate the role of Hantaan virus envelope glycoprotein in infection, a panel of monoclonal antibodies (MAbs) was examined in vitro with several serological tests and in vivo by passive transfer experiments in mice. An antigenic site, specific for the inhibition of infected cell focus was detected with the focus inhibition neutralization test (FINT), in addition to the neutralization related antigenic sites, which were revealed by the ordinary focus reduction neutralization test (FRNT). Suckling mice were given the MAbs by passive transfer followed by lethal Hantaan virus challenge. All neutralizing MAbs detected by either FRNT or FINT protected all mice from lethal infection, confirming the importance of the antigenic sites as a protective antigen. Mice given non-neutralizing MAbs by passive transfer, however, began to die earlier than the control group; mean time to death (18.2±2.1 to 21.5±2.8 days) being significantly shorter than that of the control group (25.8±1.8, p<0.01, Mann-Whitney,U probability test). Virus titers in brains of mice which died early, were about 10 times higher than those of control mice. These results indicated the early death phenomenon of mice which was mediated by the antivirus antibody.

Protective immunity of Hantaan virus nucleocapsid and envelope protein studied using baculovirus-expressed proteins

SummaryRecombinant Hantaan virus nucleocapsid protein (rNP) and recombinant envelope (rEnv) proteins were prepared using a baculovirus expression system to examine the role of Hantaan virus structural proteins in protective immunity. Passive transfer of spleen cells from mice immunized with rNP conferred partial protection or prolongation of time to death from fatal Hantaan virus infection in suckling mice which were challenged with Hantaan virus at 40 LD50 (survival rate: 43%) or 4 LD50 (survival rate: 43%). The T cell-enriched fraction protected one mouse from lethal infection but the B cell-enriched fraction had no such effect on fatal HTN infection. The protective effects of the antibody against HTN challenge were examined by passive immunization. The monoclonal antibody ECO 2 directed to NP also conferred partial survival and significant difference in time to death. Although rEnv antigen failed to induce neutralizing antibody, both immune spleen cells and immune serum to rEnv conferred partial protection upon suckling mice. These results indicate that both nucleocapsid and envelope proteins of Hantaan virus were responsible for induction of cell mediated protective immunity. Vero E 6 cells infected with Hantaan virus expressed envelope protein on the surface, as determined by flow cytometry. However, there was only negligible expression of nucleocapsid protein.

Use of Vesicular Stomatitis Virus Pseudotypes Bearing Hantaan or Seoul Virus Envelope Proteins in a Rapid and Safe Neutralization Test

ABSTRACT A vesicular stomatitis virus (VSV) pseudotype bearing hantavirus envelope glycoproteins was produced and used in a neutralization test as a substitute for native hantavirus. The recombinant VSV, in which the enveloped protein gene (G) was replaced by the green fluorescent protein gene and complemented with G protein expressed in trans (VSVΔG*G), was kindly provided by M. A. Whitt. 293T cells were transfected with plasmids for the expression of envelope glycoproteins (G1 and G2) of HTNV or SEOV and were then infected with VSVΔG*G. Pseudotype VSV with the Hantaan (VSVΔG*-HTN) or Seoul (VSVΔG*-SEO) envelope glycoproteins were harvested from the culture supernatant. The number of infectious units (IU) of the pseudotype VSVs ranged from 105 to 106/ml. The infectivity of VSVΔG*-HTN and VSVΔG*-SEO was neutralized with monoclonal antibodies, immune rabbit sera, and sera from patients with hemorrhagic fever with renal syndrome, and the neutralizing titers were similar to those obtained with native hantaviruses. These results show that VSVΔG*-HTN and -SEO can be used as a rapid, specific, and safe neutralization test for detecting hantavirus-neutralizing antibodies as an effective substitute for the use of native hantaviruses. Furthermore, the IU of VSVΔG*-HTN and -SEO did not decrease by more than 10-fold when stored at 4°C for up to 30 days. The stability of the pseudotype viruses allows distribution of the material to remote areas by using conventional cooling boxes for use as a diagnostic reagent.

Pathogenicity of Hantaan Virus in Newborn Mice: Genetic Reassortant Study Demonstrating that a Single Amino Acid Change in Glycoprotein G1 Is Related to Virulence

ABSTRACT Two Hantaan virus strains, clone 1 (cl-1), which is virulent in newborn mice, and its attenuated mutant (mu11E10), were used to examine the pathogenesis of Hantaan virus infection in a mouse model and identify virus factors relating to virulence. After subcutaneous inoculation of newborn BALB/c mice, cl-1 caused fatal disease with high viral multiplication in peripheral organs, but mu11E10 produced nonfatal infection with a low level of virus multiplication. Intracerebral inoculation of either strain caused fatal disease. Histopathological changes in the dead animals were prominent in the brain, indicating that the brain is the target organ and produces the fatal outcome. These results indicate that mu11E10 has a generally less virulent phenotype, and because of decreased multiplication in peripheral tissues, neuroinvasiveness is also decreased. An experiment with genetic reassortant viruses showed that in newborn mice the M segment is the most related to virulence and the L segment is partly related. Sequence comparison detected a single deduced amino acid change (cl-1 Ile to mu11E10 Thr) at amino acid number 515 in glycoprotein G1. One nucleotide change, but no amino acid substitution, was observed in the noncoding region of the L segment. In mouse brain microvascular endothelial cells in vitro, viruses possessing a cl-1-derived M segment grew more rapidly than viruses containing a mu11E10-derived M segment. These results suggest that the single amino acid change in the glycoprotein alters peripheral growth, which affects invasion of the central nervous system in mice.

Extensive Host Sharing of Central European Tula Virus

ABSTRACT To examine the host association of Tula virus (TULV), a hantavirus present in large parts of Europe, we investigated a total of 791 rodents representing 469 Microtus arvalis and 322 Microtus agrestis animals from northeast, northwest, and southeast Germany, including geographical regions with sympatric occurrence of both vole species, for the presence of TULV infections. Based on serological investigation, reverse transcriptase PCR, and subsequent sequence analysis of partial small (S) and medium (M) segments, we herein show that TULV is carried not only by its commonly known host M. arvalis but also frequently by M. agrestis in different regions of Germany for a prolonged time period. At one trapping site, TULV was exclusively detected in M. agrestis, suggesting an isolated transmission cycle in this rodent reservoir separate from spillover infections of TULV-carrying M. arvalis. Phylogenetic analysis of the S and M segment sequences demonstrated geographical clustering of the TULV sequences irrespective of the host, M. arvalis or M. agrestis. The novel TULV lineages from northeast, northwest, and southeast Germany described here are clearly separated from each other and from other German, European, or Asian lineages, suggesting their stable geographical localization and fast sequence evolution. In conclusion, these results demonstrate that TULV represents a promiscuous hantavirus with a large panel of susceptible hosts. In addition, this may suggest an alternative evolution mode, other than a strict coevolution, for this virus in its Microtus hosts, which should be proven in further large-scale investigations on sympatric Microtus hosts.

In vitro antiviral activity of lactoferrin and ribavirin upon hantavirus

Summary. Bovine lactoferrin (LF) and ribavirin (Rbv) were tested as antiviral agents against Seoul type hantavirus (SR-11 strain) in vitro. Hantaviral foci number in Vero E6 cells infected with SR-11 was reduced with LF treatment by 5 days post infection to obtain a 50% effective dose (ED50) of 2500 μg/ml, while pretreatment with LF was highly efficacious having an ED50 of 39 μg/ml. Conversely, 1 h pretreatment with Rbv revealed no inhibition of viral focus formation but could significantly reduce the number of viral foci (ED50: 10 μg/ml) when used from the time of viral infection. One hour pre-treatment of the cell monolayer with LF and subsequent addition of Rbv revealed a synergistic anti-hantaviral effect against SR-11, <20 FFU/ml as compared to 105 foci/ml in the control. One hour treatment of SR-11 with LF prior to cell inoculation gave an ED50 of 312.5 μg/ml. Whereas, washing the LF-pretreated cell monolayer with PBS demonstrated minimal focus reduction, suggesting LF lightly adheres to cells. These results indicate that LF has anti-hantaviral activity in vitro and inhibition of virus adsorption to cells which play an important role in revealing the anti-hantaviral activity of LF. This paper reports for the first time the anti-hantaviral effect of LF.

Mouse Mx2 protein inhibits hantavirus but not influenza virus replication.

Summary. The antiviral potential of Mx2 protein remains unknown, because the Mx2 gene in commonly used strains of laboratory mice is nonfunctional. Our previous study showed that functional Mx2 protein in some feral-origin strains was induced upon interferon treatment, was localized in the cytoplasm, and inhibited vesicular stomatitis virus replication. In the present study, we have demonstrated that the embryonic fibroblastic cells from a feral-origin strain (SPR) expressed 74 kDa Mx2 protein, which prevented the accumulation of viral transcripts and proteins of hantaviruses when the Mx2 gene was constitutively expressed in transfected Vero cells. Furthermore, the cells showed significantly lower titers of the virus than control cells. In contrast, influenza virus replication was not affected by the expression of Mx2 protein in the Vero cells.