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Oryzacystatin (OC) is the first-described cystatin originating from rice seed; it consists of two molecular species, OC-I and OC-II, which have antiviral action against poliovirus in vitro (H. Kondo, S. Ijiri, K. Abe, H. Maeda, and S. Arai, FEBS Lett. 299:48-50, 1992). In the experiments reported here, we investigated the effects of OC-I and OC-II on the replication of herpes simplex virus type 1 (HSV-1) in vitro and in vivo. HSV-1 was inoculated onto monolayers of monkey kidney epithelial cells (CV-1 cells) at a multiplicity of infection of 0.1 PFU per cell. After adsorption of the virus onto cells, the cultures were incubated in the presence of either OC-I or OC-II in the concentration range of 1.0 to 300 microM, and the supernatant virus yield was quantitated at 24 h. The effective concentration for 90% inhibition of HSV-1 was 14.8 microM, while a cytotoxic effect on CV-1 cells without infection of HSV-1 was not observed below 500 microM OC-I. Therefore, the apparent in vitro chemotherapeutic index was estimated to be more than 33. In the mouse model of HSV-1-induced keratitis and encephalopathy, topical administration of OC-I to the mouse cornea produced a significant decrease in virus production in the cornea (mean virus yields: 3.11 log10 PFU in the treated group and 4.37 log10 PFU in the control group) and significant improvement in survival rates (P = 0.01). The in vivo antiherpetic effect of OC-I was comparable to that of acyclovir, indicating that topical treatment of HSV-1 infection in humans with OC-I might be possible. Our data also suggest the importance of some thiol proteinases, which may be derived from either the host's cells or HSV-1, during the replication process of HSV-1.

SUMMARYExposure of influenza B virus-infected MDCK cells to chloroquine at the time of infection resulted in significant inhibition of infection. The appearance of input virus in the intracellular vesicles was not affected in the presence of the drug, but primary transcription of the virus genome did not occur. Chloroquine caused a rapid rise in the pH inside the lysosomes of MDCK ceils, to 6.5 from the physiological pH 5.6. In contrast, exposure of infected cells incubated in acidic medium (pH 6.0) to chloroquine did not cause an increase in lysosomal pH and this low pH treatment during the chloroquine-sensitive phase was followed by virus production. Influenza B virus induced haemolysis of chick erythrocytes at low pH values (5-0 to 5.9) which was associated with cell--cell membrane fusion. It is likely that chloroquine prevents the uncoating of influenza B virus by increasing the lysosomal pH above the critical value required for inducing fusion between the virus envelope and the lysosomal membrane.

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SUMMARYSimultaneous infection of MDCK cells with influenza viruses A/WSN and B/Kanagawa resulted in mutual interference with virus protein synthesis and in significant suppression of A/WSN growth. When infection by one virus preceded the other by 1 or 2 h, growth of the superinfecting virus was selectively inhibited at the level of transcription. Interference by the pre-infecting virus was strongly dependent on the expression of the viral genome but not on haemagglutinin activity. When the replication of both virus types was restricted to primary transcription by cycloheximide, the only translation products following removal of the drug were those of the preinfecting virus. This result was not affected by blocking secondary transcription by actinomycin D. These findings suggest that intertypic interference occurs at the level of primary transcription. This concept was supported further by the observation that a ts mutant of A/WSN (ts-65) with a defect in primary transcription interfered only with superinfection by B/Kanagawa at the permissive temperature.

Experimental infection with HVJ (haemagglutinating virus of Japan-the Sendai strain of parainfluenza 1 virus) in mice was studied. Aerosol infection of newborn mice with the wild-type virus (HVJ-W) retarded the development of body weight and killed the animals within a few weeks. Large amounts of virus were isolated from both the lungs and the nasal turbinates of infected mice. In contrast, newborn mice exposed by inhalation to a temperature-sensitive (ts) mutant (HVJ-pB) derived from an HVJ carrier culture showed no clinical signs and grew equally well as mock-infected animals. No infectious virus could be recovered from the lungs although the ts mutant grew to moderate titre in the nasal turbinates. The prior inoculation of newborn mice with the ts mutant virus induced a state of significant resistance to subsequent challenge with the virulent wild-type virus. No replication of challenge virus in both lungs and nasal turbinates could be detected and the animals were protected a lethal infection. It is suggested that an avirulent temperature-sensitive mutant which has lost the capacity to replicate in the lower respiratory tract but is still capable of multiplying in the nasal turbinates may be a promising candidate for use in live vaccines especially against the infectious disease of the lower respiratory tract.

Based on the information that high salt inhibits the initiation of cellular mRNA translation which depends on the function of the 5'-terminal structure of mRNA, we compared the effect of high salt on translation of host cellular mRNAs and influenza viral mRNAs, both of which are of 5 '-terminal structure. Brief exposure of influenza B virus-infected MDCK cells to high salt medium resulted in a dosedependent inhibition of viral polypeptide synthesis as well as of cellular polypeptide synthesis, but it had less effect on synthesis of viral polypeptides, particularly nonstructural protein (NS). Under these conditions the Nar content of the infected cells was significantly increased. A similar salt effect on in vitro translation of viral and cellular mRNAs extracted from infected cells was also observed. There was no significant difference in sensitivity to hypertonic block of in vivo translation of influenza viral mRNAs and vesicular stomatitis virus mRNAs, the latter of which possess a virus-directed structure at the 5 '-terminus.

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