[Differences in susceptibility to peripheral infection with Japanese encephalitis virus among inbred strains of mouse].

We compared susceptibility of inbred mouse strains against intracerebral as well as peripheral challenge of a flavivirus, Japanese encephalitis (JE) virus, and the results were summarized as follows: (1) Seven inbred mouse strains (C3H/He, C57BL/6, BALB/c, AKR/N, NC, NZB, DBA/2) could be classified into following 3 groups by their mortality and infection rate when they were subcutaneously challenged with a wild JE virus strain isolated from field-caught mosquitoes at a single passage in mouse (B18AM, B-1): (1) high mortality and high infection rate (C3H/He, C57BL/6, AKR/N), (2) low mortality but high infection rate (BALB/c, NC, NZB), and (3) low mortality and low infection rate (DBA/2). The DBA/2 strain showed lowest mortality and infection rate with statistically significant difference with other strains. (2) Three inbred mouse strains (C3H/He, C57BL/6, BALB/c) were peripherally challenged with following 6 different wild strains of JE virus: mosquito origin after a single passage in mouse (B-1); mosquito origin after 30 passages in mice by subcutaneous inoculations (BP-30), mosquito origin after 30 passages in mice by intracerebral inoculations (BB-30); human origin after a single passage in mouse (JaNH180,J-1); human origin after 30 passages in mice by subcutaneous inoculations (JP-30); human origin after 30 passages in mice by intracerebral inoculations (JB-30). Survival of these inbred mice showed curves, with statistical difference among mouse strains. Anti-JE ELISA antibody titers in survived mice were lowest in BALB/c among 3 inbred mouse strains for all JE virus strains used to the challenge experiment. The result indicated that BALB/c would be a suitable host to study inapparent JE virus infection which is frequent among humans exposed to JE virus. (3) Male mice showed higher mortality than females when 4 weeks old mice were peripherally challenged by JE virus, and the difference was statistically significant. The results as a whole indicated that the susceptibility of mice to JE virus is under the control of genetic factors.

Radiation inactivation analysis of fusion and hemolysis by vesicular stomatitis virus.

Radiation inactivation analysis was used to determine the size of the functional unit responsible for fusion of vesicular stomatitis virus (VSV) with cardiolipin or phosphatidylcholine-phosphatidylethanolamine (1:1) liposomes, and for VSV-induced hemolysis. When radiation-insensitive background values were subtracted, the calculated functional units for all three activities were similar, ranging from 866 to 957 kDa, equivalent to about 15 G protein molecules. This is in striking contrast to results of similar studies with influenza and Sendai viruses, in which the functional unit corresponded in size to a single fusion protein monomer, and suggests that VSV fusion may occur by a different mechanism.

Fusion of a Sendai mutant deficient in HN protein (ts271) with cardiolipin liposomes.

Sendai mutant ts271 contains less than 5% of the amount of HN glycoprotein found in wild-type Sendai. Fusion of this mutant with cardiolipin liposomes revealed no differences from the wild-type virus with regard to specific activity, pH dependence, or radiation inactivation. Target sizes of both mutant and wild-type viral proteins were determined by the radiation-induced disappearance of each band from an SDS-polyacrylamide gel and no differences were found. Of the viral proteins, only F had a target size corresponding to the monomer molecular weight, ca. 60 kDa, identical to the minimum unit previously determined by functional assay for Sendai virus-erythrocyte membrane fusion (K. Bundo-Morita, S. Gibson, and J. Lenard, Biochemistry 26, 6223-6227 (1987)). This provides additional evidence that F alone is the active protein mediating Sendai-erythrocyte fusion. It is concluded that the HN protein is unlikely to mediate any fusion reactions of the intact virions, either with biological membranes or with cardiolipin liposomes.

Estimation by radiation inactivation of the size of functional units governing Sendai and influenza virus fusion.

The target sizes associated with fusion and hemolysis carried out by Sendai virus envelope glycoproteins were determined by radiation inactivation analysis. The target size for influenza virus mediated fusion with erythrocyte ghosts at pH 5.0 was also determined for comparison; a value of 57 +/- 15 kDa was found, indistinguishable from that reported previously for influenza-mediated fusion of cardiolipin liposomes [Gibson, S., Jung, C. Y., Takahashi, M., & Lenard, J. (1986) Biochemistry 25, 6264-6268]. Sendai-mediated fusion with erythrocyte ghosts at pH 7.0 was likewise inactivated exponentially with increasing radiation dose, yielding a target size of 60 +/- 6 kDa, a value consistent with the molecular weight of a single F-protein molecule. The inactivation curve for Sendai-mediated fusion with cardiolipin liposomes at pH 7.0, however, was more complex. Assuming a "multiple target-single hit" model, the target consisted of 2-3 units of ca. 60 kDa each. A similar target was seen if the liposomes contained 10% gangliosides or if the reaction was measured at pH 5.0, suggesting that fusion occurred by the same mechanism at high and low pH. A target size of 261 +/- 48 kDa was found for Sendai-induced hemolysis, in contrast with influenza, which had a more complex target size for this activity (Gibson et al., 1986). Sendai virus fusion thus occurs by different mechanisms depending upon the nature of the target membrane, since it is mediated by different functional units. Hemolysis is mediated by a functional unit different from that associated with erythrocyte ghost fusion or with cardiolipin liposome fusion.