Ikuo Takashima

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.

Mortality following peripheral infection with Tick-borne encephalitis virus results from a combination of central nervous system pathology, systemic inflammatory and stress responses

Tick-borne encephalitis virus (TBEV) induces acute central nervous system (CNS) disease in humans. In this study, we investigate the pathogenetic mechanisms that correlate with fatal infection with TBEV in a mouse model. Following subcutaneous infection with high challenge doses (>10(7) PFU), mice started to die early (8 days) and mortality rates reached >80%. These doses induced acute and widespread infection of the CNS. On the other hand, following subcutaneous infection with low challenge doses (10(2)-10(6) PFU), mice started to die late (11 days) and approximately one half of the mice survived but exhibited degrees of encephalitis similar to dying mice. However, low dose dying mice exhibited severe systemic stress response, and increased levels of TNF-alpha compared with recovering mice. We therefore conclude that in addition to the development of CNS disease, systemic inflammatory and stress responses contribute to induce a fatal infection following subcutaneous infection of mice with TBEV.

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.

Phylogenetic and virulence analysis of tick-borne encephalitis viruses from Japan and far-eastern Russia

We have previously reported that tick-borne encephalitis (TBE) is endemic in a specific area of Hokkaido, Japan. In Oshima, the southern part of Hokkaido, TBE virus was isolated from sentinel dogs, ticks and rodents in 1995 and 1996. To identify when these TBE viruses emerged in Hokkaido, the times of divergence of TBE virus strains isolated in Oshima and far-eastern Russia were estimated. TBE virus was isolated in Khabarovsk in 1998 and the nucleotide sequences of viral envelope protein genes of isolates from Oshima and Khabarovsk were compared. From the synonymous substitution rate of these virus strains, the lineage divergence time of these TBE virus strains was predicted phylogenetically to be about 260-430 years ago. Furthermore, the virulence of TBE virus isolates from Oshima and Khabarovsk were compared in a mouse model. The results showed that the isolates possessed very similar virulence in mice. This report provides evidence that the Oshima strains of TBE virus in Hokkaido emerged from far-eastern Russia a few hundred years ago and this explains why these strains possess virulence similar to the TBE viruses isolated in Russia.

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.

Protection against tick-borne encephalitis virus isolated in Japan by active and passive immunization.

In order to establish a firm preventive measure for tick-borne encephalitis (TBE) in Japan, we evaluated the immune response of European vaccine against Japanese TBE virus strain (Oshima 5-10) for man and mouse. Furthermore, the efficacy of pre- and post-exposure protection by a polyclonal rabbit anti-TBE virus serum was examined in the mouse model. 80% of vaccinees seroconverted against Oshima 5-10 strain after the 2nd immunization of vaccine and the remaining 20% seroconverted after the 3rd immunization. Two persons with pre-existing anti-Japanese encephalitis virus (JEV) antibodies showed low immune responses against TBE virus. In mouse vaccination and challenge tests, efficient protection was observed in mice challenged with lethal doses of Oshima 5-10 strain as well as those observed in mice with the Western subtype and the Far Eastern subtype of TBE strains. Pre-exposure treatment with rabbit anti-TBE virus serum provided complete protection against lethal challenge with Oshima 5-10 strain. For post-exposure treatment with the antibody, significant protection was observed when mice were treated 24 h after virus challenge, whereas it was not observed 48 h after virus challenge. reserved.

Involvement of the JNK-like protein of the Aedes albopictus mosquito cell line, C6/36, in phagocytosis, endocytosis and infection of West Nile virus

We recently cloned a c‐Jun amino‐terminal kinase (JNK) sequence from the C6/36 cell line, derived from the mosquito Aedes albopictus. We showed that SP600125, an inhibitor of JNK proteins, inhibits phagocytosis by C6/36 cells, suggesting that the JNK‐like protein regulates phagocytosis. Here, we show that C6/36 cells constitutively express low levels of mRNA encoding the antibacterial peptides, cecropin and defensin, but that these mRNAs were up‐regulated upon stimulation by lipopolysaccharide (LPS). Thus, the C6/36 cells have properties similar to those of mammalian macrophages. To characterize further the functional properties of C6/36 cells, we have assayed the role of the JNK‐like protein in phagocytosis, endocytosis, and viral infection. C6/36 cells phagocytosed bacteria and artificial beads, and this was only slightly up‐regulated following LPS stimulation, suggesting that newly stimulated JNK‐like protein was not necessary for phagocytosis. SP600125 inhibited the acidification of intracellular compartments, including those involved in the endocytic pathway. Pretreatment of C6/36 cells with SP600125 or bafilomycin A1, but not cytochalasin D, inhibited the entry of West Nile virus (WNV), suggesting that WNV is internalized mainly by endocytosis, and that the JNK signalling pathway is important for endocytic entry. These findings indicate that the JNK‐like protein regulates basic physiological functions, including phagocytosis and endocytosis and infection of WNV.

Cell fusion by haemorrhagic fever with renal syndrome (HFRS) viruses and its application for titration of virus infectivity and neutralizing antibody

SummaryHaemorrhagic fever with renal syndrome viruses, are members of the family Bunyaviridae. They cause cell to cell fusion from within under acidic conditions. This phenomenon was found to occur under a pH range of between 4.9 to 6.3 for all the viruses examined. The pH range which causes cell fusion was similar to that reported for the La Crosse virus of the Bunyaviridae, hence indicating that this property is a common biological characteristic among this family of viruses.Titration of virus infectivity and neutralizing antibody was done by counting the number of fused cell foci produced in infected Vero cell monolayers after low pH treatment. This method was simpler and more rapid than the ordinary plaque formation method or that of counting infected cell foci by IFA or immunoenzyme assay. In addition, this method may also be applicable in the detection of other enveloped viruses which do not cause a typical cytopathic effect.

Genetic diversities of hantaviruses among rodents in Hokkaido, Japan and Far East Russia

Seroepizootiologic surveys among wild rodents were carried out in Japan and Far East Russia in 1995 and 1996. Seropositive animals were only identified in Clethrionomys rufocanus (23/134) in Hokkaido, Japan. On the other hand, seropositives were identified in C. rufocanus (1/8), Apodemus agrarius (2/66), Apodemus spp. (2/26) and Microtus fortis (3/22) in Vladivostok, Far East Russia. Total RNA was isolated from lungs of seropositive animals and the S genome segments were amplified by PCR, cloned and sequenced. The S and M genomes of hantavirus, derived from Japanese C. rufocanus (Tobetsu genotype), were most closely related with Puumala viruses (76-79% nucleotide and 95% amino acid identities for S genome, 70-78% nucleotide and 87-92% amino acid identities for M genome). The recombinant nucleocapsid protein of Tobetsu genotype was antigenically quite similar with that of Sotkamo. These suggest that the virus endemic in Japanese C. rufocanus belongs to Puumala virus. Phylogenetic analysis indicates that the genotype forms a distinct lineage within Puumala viruses. Partial S segment (1-1251 nt), derived from seropositive M. fortis in Vladivostok, was sequenced and analyzed. The S genome segment, which was designated Vladivostok genotype, was most closely related with Khabarovsk virus (79% nucleotide and 90% amino acid identities) which was isolated from M. fortis.

Characteristics of passive immunity against hantavirus infection in rats

SummaryThe protective effects of passively administered antibodies against hantavirus infection were studied in newborn rats. Death as well as infection were completely prevented from intraperitoneal challenge of strain SR-11 (SR) (2×103 FFU, 102.1 LD50), in newborn rats which received 0.1 ml of anti-SR rat serum (neutralizing antibody titer, 1:640) 4hr before the virus challenge. In these rats, no virus was detected in the peritoneal macrophages, lung, kidney, and brain. The immune serum infusion before the virus challenge also conferred protection to rats against an intramuscular or subcutaneous challenge of strain SR, but did not protect the rats against intracerebral challenge of the virus. In the rats which received the immune serum after the challenge, infection was not prevented, although some of the animals were protected from the death. Virus titers in the lung, kidney, and brain of the rats were reduced by the transfer of the immune serum even as late as 72hr after the challenge. Cross-protection in the rats which received the immune serum was strong between strains SR and KI-262 within the same serotype, but very weak between strains SR and Hantaan 76–118 of different serotypes.