Norio Hirano

Analysis of genomic and intracellular viral RNAs of small plaque mutants of mouse hepatitis virus, JHM strain

Abstract The genomic RNA and intracellular RNA of mouse hepatitis virus, strain JHM (MHV-JHM) and two plaque mutants (la and 2c), which have been isolated from a persistently infected culture (JHM-CC), have been analyzed by T1-resistant oligonucleotide finger-printing. The genomic RNA of the virus population (JHM-CC virus) released from different passage levels of the same persistent infection has also been analyzed. The analysis shows the locations within the genomic and intracellular RNAs of more than 45 T1-resistant oligonucleotides and confirm earlier studies (J. L. Leibowitz, K. C. Wilhelmsen, and C. W. Bond (1981), Virology 114, 39–51), showing that the six subgenomic RNAs of MHV-JHM form a 3′ coterminal nested set which extends for different lengths in a 5′ direction. The analysis also identifies in each subgenomic RNA those large T1 oligonucleotides derived from noncontiguous regions of the genome during mRNA synthesis. Two important conclusions can be reached from analysis of the mutant viruses. First, the virus population released from the persistent infection represents a fairly constant mixture of viruses, and the fluctuating emergence of variants as predominant species in the culture does not occur. Second, the data indicate that for particular intracellular RNAs of mutant viruses the sequence rearrangements occurring during subgenomic mRNA synthesis are different from those in the corresponding intracellular RNA of wild-type virus. The result may indicate a potential flexibility in the leader/body fusion process that has not been previously recognized.

Comparison of mouse hepatitis virus strains for pathogenicity in weanling mice infected by various routes

SummaryThe pathogenicity for mice of nine strains of mouse hepatitis virus was studied in mice free from the virus by the intracerebral, intraperitoneal, intravenous and intranasal routes of inoculation.

Persistent Infection with Mouse Hepatitis Virus, JHM Strain in DBT Cell Culture

After inoculation with JHM strain into DBT cell monolayers, a persistently infected DBT cell culture was established without producing typical cytopathic changes after about 15th passages. By immunofluorescence virus specific antigen was demonstrated in 10 to 15% DBT cells. This persistently infected culture (JHM-CC) was resistant to superinfection with parental JHM, but such resistance was not shown against vesicular stomatitis virus. JHM-CC virus produced small plaques on DBT cell monolayers. Temperature sensitive (TS) mutant, defective interfering (DI) particle or interferon was not detected in the JHM-CC. To intracerebral inoculation with JHM-CC virus, cortisone treated ICR mice survived without showing clinical signs, however, demyelinating lesions were produced in the brain and spinal cord of them.

Replication and plaque formation of swine hemagglutinating encephalomyelitis virus (67N) in swine cell line, SK-K culture.

Abstract Swine hemagglutinating encephalomyelitis virus (HEV), 67N strain, adapted to suckling mouse brain, grew readily in a porcine cell line, SK-K cell culture with cytopathic effect (CPE) consisting of syncytium formation and detachment of fused cells and round cells from glass surface. After further passages in SK-K cell monolayers with undiluted culture fluid, CPE developed earlier and became complete within 48 h postinoculation (p.i.). Viral specific antigen was detected in the cytoplasm of the infected SK-K cells by indirect immunofluorescence using rabbit antiserum against the mouse-passaged virus. The SK-K-passaged virus as well as the original mouse-passaged virus formed clear plaques on SK-K cell monolayers under simple overlay medium. The plaque assay system for HEV 67N was established by studying various factors influencing the plaque formation in the SK-K cell cultures. By this system more than 106 PFU/0.2 ml of the virus yield was detected in the fluid phase of the infected cultures at 48 h p.i. The SK-K-passaged virus caused fatal infection in 4-week-old mice by intracerebral inoculation, but was inhibited by rabbit antiserum against the mouse-passaged virus. Plaque formation and hemagglutinating activity of the virus were specifically inhibited by antisera against the mouse-passaged and SK-K-passaged 67N virus.

Replication of rat coronavirus in a rat cell line, LBC

SummaryRat coronavirus readily propagated and induced marked cytopathic effect in a rat cell line, LBC cell culture, which provided a sensitive, practical assay system for viral infectivity and neutralizing antibody, and a satisfactory source of the virus.

Replication of sialodacryoadenitis virus of rat in LBC cell culture. Brief report.

SummarySialodacryoadenitis virus of rat readily propagated and induced marked cytopathic effect in a rat cell line, LBC cell culture, which provides a sensitive, practical assay system for viral infectivity and neutralizing antibody, and a satisfactory source of the virus.

Neuronal Spread of Swine Hemagglutinating Encephalomyelitis Virus (HEV) 67N Strain in 4-Week-Old Rats

The HEV 67N strain causes encephalomyelitis or vomiting and wasting syndrome in piglets 1,2. In experimental infection of piglets, the virus spreads along the nerve pathways to the central nervous system (CNS), and is restricted to the neurons. In our experimental studies of HEV 67N strain, the virus produced encephalitis in mice when inoculated by several routes, and propagated mainly in the neurons in the CNS 4. However, 20-day-old or older mice were resistant to the virus inoculated by intravenous (i.v.), intraperitoneal (i.p.) or subcutaneous (s.c.) route. In contrast, 4-week-old rats died of encephalitis after i.v., i.p. and s.c. as well as intracerebral (i.c.) and intranasal (i.n.) inoculation. However, when rats were inoculated by s.c. route, they died a few days earlier than those by i.p. and i.v. routes, suggesting that the virus might be spread to the CNS by neural routes rather than blood stream. To see the virus spread from the peripheral nerve to the brain, the virus was directly inoculated in to the sciatic nerve of rats. These studies demonstrated that HEV spread from the sciatic nerve to the brain by the neural route, and that persistent infection of HEV was established in rats6.

Neurotropic virus tracing suggests a membranous-coating-mediated mechanism for transsynaptic communication

Swine hemagglutinating encephalomyelitis virus (HEV) has been shown to have a capability to propagate via neural circuits to the central nervous system after peripheral inoculation, resulting in acute deadly encephalomyelitis in natural host piglets as well as in experimental younger rodents. This study has systematically examined the assembly and dissemination of HEV 67N in the primary motor cortex of infected rats and provides additional evidence indicating that membranous‐coating‐mediated endo‐/exocytosis can be used by HEV for its transsynaptic transfer. In addition, our results suggested that this transsynaptic pathway could adapted for larger granular materials, such as viruses. These findings should help in understanding the mechanisms underlying coronavirus infections as well as the intercellular exchanges occurring at the synaptic junctions. J. Comp. Neurol. 521:203–212, 2013.

Spread of Swine Hemagglutinating Encephalomyelitis Virus From Peripheral Nerves to the CNS

Swine hemagglutinating encephalomyelitis virus (HEV) strain 67N was inoculated into the sciatic nerve or the right leg crural muscle of rats. In both cases, the virus was isolated first from the caudal half of the spinal cord on day 2 after inoculation, and from the rostral half of the spinal cord and the brain on day 3. The virus titers in the brain reached a maximum when the infected rats developed CNS symptoms on day 5. Using confocal laser scanning microscope, fluorescent positive cells were first found in the lumbar dorsal root ganglion (DRG) and spinal cord ipsilateral of the inoculated leg on day 3. Antigen positive neurons were found bilaterally in the lumbar DRG and spinal cord on day 4. On day 5 specific fluorescence was observed in the neurons of the cerebral cortex, hippocampus, brainstem and Purkinje cells in the cerebellum.

The route of transmission of hemagglutinating encephalomyelitis virus (HEV) 67N strain in 4-week-old rats.

Four-week-old Wistar rats were inoculated with HEV by different routes. Animals died of encephalitis after intraperitoneal (i.p.), subcutaneous (s.c.) and intravenous (i.v.) as well as intracerebral (i.c.) and intranasal (i.n.) inoculation. However when inoculated subcutaneously, rats died a few days earlier than those inoculated i.p. and i.v., suggesting that the virus might be transmitted to the central nervous system (CNS) by the neuronal route rather than by blood stream. Rats which were inoculated subcutaneously at the site of the neck (group A) began to die on day 4 p.i., a few days earlier than animals inoculated in the foot pad of the right leg (group B). On day 2 and 3 after inoculation, the virus titer in the brain was higher in group A, but group B animals showed higher virus titers in the lumber region of spinal cord than group A animals. In order to follow the virus spread from the peripheral nerve to the brain, the virus was inoculated into the sciatic nerve of rats. The inoculated rats developed clinical signs on day 4 and began to die on day 6. On day 2, virus was detected in the posterior half of the spinal cord and migrated toward the anterior half and in the brain where it was present on day 3. The highest virus titers in the brain were recorded on day 4 to 6, meanwhile the virus titers in the spinal cord tend to decrease. By immunohistochemical study, antigen positive neurons were found in the spinal cord and brain on day 4.(ABSTRACT TRUNCATED AT 250 WORDS)