Epizootiological study of tick-borne encephalitis virus infection in Japan.

Tick-borne encephalitis virus (TBEV) is a zoonotic agent causing severe encephalitis in humans. Rodent species that are potential hosts for TBEV are widely distributed in various regions in Japan. In this study, we carried out large-scale epizootiological surveys in rodents from various areas of Japan. A total of 931 rodent and insectivore sera were collected from field surveys. Rodents seropositive for TBEV were found in Shimane Prefecture in Honshu and in several areas of Hokkaido Prefecture. These results emphasize the need for further epizootiological and epidemiological research of TBEV and preventive measures for emerging tick-borne encephalitis in Japan.

The binding potential of commercial antibody conjugates with sera of various small terrestrial mammals.

Infectious diseases of wild animals are of increasing importance, both from an economic viewpoint and because several of these diseases are pathogenic to man. However, serosurveys to determine the circulation of infectious organisms in wildlife are complicated by the fact that antibodies to species-specific immunoglobulins are not available for use in serological assays such as enzyme-linked immunosorbent assays (ELISAs) or immunofluorescence assays. To determine the binding potential of four commercially available antibody conjugates with the sera of wild animals, sera from 27 species of small terrestrial mammals were allowed to react with alkaline phosphatase-labelled protein A, anti-rabbit IgG, anti-mouse IgG and anti-human IgG by by the use of an ELISA. It was found that sera from some species of the order Lagomorpha bound optimally to anti-rabbit IgG, while anti-mouse IgG could be used for most species of Rodentia. For all Carnivora, Insectivora, Macroscelidea, Hyracoidea and other Rodentia, staphylococcal protein A demonstrated optimal binding. None of the sera that was tested bound to anti-human IgG. These results demonstrate that commercial conjugates can be used in serological assays in which wild animal sera are used, and should be useful for future serosurveys to determine the circulation of infectious agents in small terrestrial mammals.

Development of serological assays for Thottapalayam virus, an insectivore-borne Hantavirus.

Thottapalayam virus (TPMV), a member of the genus Hantavirus in the family Bunyaviridae, was isolated from an insectivore, Suncus murinus (musk shrew), captured in southern India in 1964. While the isolation of TPMV predates the discovery of the prototype Hantaan virus, little is known about its genetics and biology. To date, preliminary evidence suggests that TPMV differs significantly, both antigenically and genetically, from all known rodent-borne hantaviruses. However, since detailed epizootiological studies have not been conducted, it is unclear if TPMV is naturally harbored by an insectivore host or if TPMV represents a "spillover" from its natural rodent reservoir host. Moreover, to what extent TPMV causes infection and/or disease in humans is not known. To address these issues, we first studied the antigenic profile of TPMV using monoclonal antibodies against Hantaan and Seoul viruses and polyclonal immune sera against Puumala virus and TPMV. Armed with this newfound information, we developed an enzyme-linked immunosorbent assay system for the diagnosis of TPMV infections in shrews and humans, using a recombinant TPMV N antigen manipulated to have an E5/G6 epitope to be captured by monoclonal antibody clone E5/G6. Using this assay, we found anti-TPMV antibodies in sera from a patient with high fever of unknown etiology in Thailand and from two shrews captured in Indonesia. Seropositivity was verified by the indirect immunofluorescence antibody test, Western blotting analysis, and focus reduction neutralization test. Collectively, our data indicate that TPMV is harbored by Suncus murinus as its host in nature and is capable of infecting humans.

[Hemoglobins, XLIII: The primary structure of mole hemoglobin (Talpa europaea) (author's transl)]. / Hämoglobine, XLIII. Die Primärstruktur des Hämoglobins des Maulwurfs (Talpa europaea).

The hemoglobin of the european mole (Talpa europaea) has only one component. The alpha- and beta-chains were separated by chromatography on CM 52 cellulose. The primary structures of both chains were mainly established on the tryptic peptides by automatic Edman degradation. The N-terminal regions were sequenced on the chains. Large C-terminal peptides could be isolated and sequenced after acidic hydrolysis of the Asp-Pro bond (alpha: 94/95, beta: 99/100). The peptides were aligned by their homology with human alpha- and beta-chains. A comparison with human hemoglobin shows an exchange of 18 amino acid residues in the alpha-chains and of 30 in the beta-chains. The substitutions in the surroundings of the heme and the contacts between the subunits are discussed. All amino acid residues responsible for the binding of 2,3-dihosphoglycerate are present. Nevertheless a high oxygen affinity and a reduced interaction with diphosphoglycerate are found in mole hemoglobin (Jelkmann, W., Oberthür, W., Kleinschmidt, T. & Braunitzer, G. (1981) Respir. Physiol., in press). According to the sequence a relaxed structure of the central cavity between the beta-chains can be the reason for the low interaction between phosphate and protein. The oxygen affinity is not only affected by the presence of diphosphoglycerate-binding sites in hemoglobin but also by the structure in this region of the molecule.

Adaptation of hemoglobin function to subterranean life in the mole, Talpa europaea.

In order to understand the mechanism responsible for the high oxygen affinity of mole blood, we investigated in the mole. Talpa europaea, red cell parameters that determine hemoglobin function. We have found that the oxygen half saturation pressure (P50) of mole blood is 2.85 kPa (21.4 Torr) at pCO2 4.7 kPa, pH 7.4 and 37 degree C. The concentration of 2,3-diphosphoglycerate (2,3-DPG) averaged 5.3 mmol/l in red cells. In addition, we have determined P50 in hemoglobin solutions at various concentrations of 2,3-DPG at an assumed intraerythrocytic pH of 7.2 and 37 degree C. These data were used to calculate the association constants of 2,3-DPG to mole hemoglobin. P50 was 1,89 kPa (14.2 Torr) in hemoglobin solutions without 2,3-DPG. The response to 2,3-DPG was relatively low. Noteworthy, CO2 did not affect the oxygen affinity at constant pH in the presence of 2,3-DPG. Our results suggest that the high blood oxygen affinity of the mole can be attributed to a weak interaction of its hemoglobin with 2,3-DPG.

Evidence for the presence of alpha 1B-glycoprotein in mammalian sera: immunoblotting studies.

1. A monospecific antiserum to pig alpha 1B-glycoprotein (PO2) was produced in rabbits and was used to search for homologues of alpha 1B in sera of 41 mammalian species belonging to seven orders. 2. Specific reactions were detected in the sera of representatives of Insectivora, Primates, Carnivora, Proboscidea, Perissodactyla and Artiodactyla. No cross-reactions were observed in the sera of two species of Rodentia (mouse, rat). 3. Cross-reactions in the sera of Erinaceus europaeus, Homo sapiens and Macaca mulatta were rather weak; this indicates a greater structural difference between the alpha 1 B of Insectivora and Primates and that of the other mammalian orders. 4. Electrophoretic patterns of alpha 1 B were, in most cases, heterogeneous, the most heterogeneous being in ruminants. 5. Evidence was obtained that the alpha 1 B of sheep is identical with the earlier described (Juneja and Gahne (1980) Anim. Blood Grps Biochem. Genet. 11, 81-92.) polymorphic post-transferrin (Ptf).

The evolution of the thyroid hormone distributor protein transthyretin in the order insectivora, class mammalia.

Thyroid hormones are involved in the regulation of growth and metabolism in all vertebrates. Transthyretin is one of the extracellular proteins with high affinity for thyroid hormones which determine the partitioning of these hormones between extracellular compartments and intracellular lipids. During vertebrate evolution, both the tissue pattern of expression and the structure of the gene for transthyretin underwent characteristic changes. The purpose of this study was to characterize the position of Insectivora in the evolution of transthyretin in eutherians, a subclass of Mammalia. Transthyretin was identified by thyroxine binding and Western analysis in the blood of adult shrews, hedgehogs, and moles. Transthyretin is synthesized in the liver and secreted into the bloodstream, similar to the situation for other adult eutherians, birds, and diprotodont marsupials, but different from that for adult fish, amphibians, reptiles, monotremes, and Australian polyprotodont marsupials. For the characterization of the structure of the gene and the processing of mRNA for transthyretin, cDNA libraries were prepared from RNA from hedgehog and shrew livers, and full-length cDNA clones were isolated and sequenced. Sections of genomic DNA in the regions coding for the splice sites between exons 1 and 2 were synthesized by polymerase chain reaction and sequenced. The location of splicing was deduced from comparison of genomic with cDNA nucleotide sequences. Changes in the nucleotide sequence of the transthyretin gene during evolution are most pronounced in the region coding for the N-terminal region of the protein. Both the derived overall amino sequences and the N-terminal regions of the transthyretins in Insectivora were found to be very similar to those in other eutherians but differed from those found in marsupials, birds, reptiles, amphibians, and fish. Also, the pattern of transthyretin precursor mRNA splicing in Insectivora was more similar to that in other eutherians than to that in marsupials, reptiles, and birds. Thus, in contrast to the marsupials, with a different pattern of transthyretin gene expression in the evolutionarily "older" polyprotodonts compared with the evolutionarily "younger" diprotodonts, no separate lineages of transthyretin evolution could be identified in eutherians. We conclude that transthyretin gene expression in the liver of adult eutherians probably appeared before the branching of the lineages leading to modern eutherian species.