Enterotropic mouse hepatitis virus infection in nude mice

The cause of emaciation and diarrhea in athymic nude mice was found to be hyperplastic typhlocolitis resulting from infection with enterotropic mouse hepatitis virus (MHV). The disease was reproduced in experimentally-inoculated nude mice using intestinal homogenates from affected mice and cell culture-derived virus. Material derived from an experimental mouse was passed into neonatal Swiss mice and caused acute typhlocolitis. Virus failed to grow in NCTC-1469 cells and 17Cl-1 cells, which are normally permissive for MHV, but grew to low titer in a mouse rectal carcinoma cell line, CMT 93. These results show that an enterotropic strain of MHV can cause chronic enteric disease in athymic nude mice. The pattern of infection differs markedly from the more common MHV wasting syndrome in nude mice caused by non-enteric strains of MHV.     

Dynamics of viral RNA synthesis during measles virus infection

We propose a reference model of the kinetics of a viral RNA-dependent RNA polymerase (vRdRp) activities and its regulation during infection of eucaryotic cells. After measles virus infects a cell, mRNAs from all genes immediately start to accumulate linearly over the first 5 to 6 h and then exponentially until approximately 24 h. The change from a linear to an exponential accumulation correlates with de novo synthesis of vRdRp from the incoming template. Expression of the virus nucleoprotein (N) prior to infection shifts the balance in favor of replication. Conversely, inhibition of protein synthesis by cycloheximide favors the latter. The in vivo elongation speed of the viral polymerase is approximately 3 nucleotides/s. A similar profile with fivefold-slower kinetics can be obtained using a recombinant virus expressing a structurally altered polymerase. Finally, virions contain only encapsidated genomic, antigenomic, and 5'-end abortive replication fragment RNAs.     

Role of macrophages during Theiler's virus infection.

Theiler's virus, a murine picornavirus, causes a persistent infection of the central nervous system with chronic inflammation and primary demyelination. We examined the nature of infected cells at different times postinoculation (p.i.) with a combined immunocytochemistry-in situ hybridization assay. The virus was found in the gray matter of the brain, mostly in neurons, during the first week p.i. During the following weeks, the virus was present in the spinal cord, first in the gray and white matter, then exclusively in the white matter. Approximately 10% of infected cells were astrocytes at any time during the study. Infected oligodendrocytes were first noticed on day 14 p.i. and amounted to approximately 6% of infected cells. The number of infected macrophages increased with time and reached a plateau by day 21 p.i., when at least 45% of infected cells were macrophages. The role of blood-borne macrophages during infection was studied by depleting them with mannosylated liposomes containing dichloromethylene diphosphonate. The virus did not persist in the majority of the mice treated with liposomes. These mice showed only minimal mononuclear cell infiltration and no demyelination.     

Restricted viral RNA synthesis in establishment of persistent infection in Vero cells with a Sendai virus mutant

It was previously shown that a temperature-sensitive mutant of Sendai virus, ts-23, readily establishes persistent infection in Vero cells at 37 C, a permissive temperature for growth of the mutant. In the present study, it was demonstrated that the virus yield from ts-23-infected Vero cells at 37 C began to decrease 48 to 72 hr postinfection, after an initial phase of high virus production. Before the decrease in virus production, the formation of viral nucleoprotein declined, although synthesis of all species of viral protein continued. It was suggested that the limited formation of viral nucleoprotein and the decrease in virus production were due to the restriction of viral RNA synthesis which began to occur early after infection in ts-23-infected cells at 37 C. The mutant has a temperature-sensitive defect in RNA polymerase activity and the temperature 37 C, used for establishment of persistent infection, would be a semi-permissive temperature for the RNA polymerase activity of the mutant. The ts-23 mutant interfered with the replication of the parental wild virus in Vero cells at 37 C.     

Influence of the Theiler's virus L* protein on macrophage infection, viral persistence, and neurovirulence

The genome of picornaviruses contains a large open reading frame (ORF) translated as a precursor polypeptide that is processed to yield all the proteins necessary for the viral life cycle. In persistent but not in neurovirulent strains of Theiler's virus, an overlapping ORF encodes an additional 18-kDa protein called L*. We confirmed previous work showing that the L* ORF of persistent strains facilitates the infection of macrophage cell lines, and we present evidence that this effect is due to the L* protein itself rather than to competition for the translation of the two overlapping ORFs. The introduction of an AUG codon to restore the L* ORF of the neurovirulent GDVII strain also enhanced the infection of macrophages, in spite of the divergent evolution of this protein. The presence or the absence of the L* AUG initiation codon had only a weak influence on the neurovirulence of the GDVII strain and on the persistence of the DA1 strain. The results obtained with DA1 in vivo contrast with the results reported previously for DAFL3, another molecular clone of the same virus strain, where the AUG-to-ACG mutation of the L* initiation codon totally blocked viral persistence (G. D. Ghadge, L. Ma, S. Sato, J. Kim, and R. P. Roos, J. Virol. 72:8605-8612, 1998). Thus, a factor that is critical for the persistence of a given clone of Theiler's virus is dispensable for the persistence of a closely related clone, indicating that different adjustments in the expression of persistence determinants occur in related viral strains.     

Persistent infection with mouse hepatitis virus of low virulence in nude mice.

A persisting type of infection with wasting syndrome was established in congenitally athymic nude mice after intraperitoneal inoculation with a mouse hepatitis virus which was not fully pathogenic for heterozygous haired littermates. From the liver, spleen, lymph nodes, and brain of most infected nude mice, the virus was detected at high titers during aperiod from 6 to 35 days postinfection, occurrence of degenerative and necrotic lesions being correlated with virus titers in these organs. The titer of serum neutralizing antibody remained undetectable or very low in most diseases nude mice, whereas some animals resisting the infection could produce antibody at a later stage. In heterozygous haired mice, some lesions were detectable at a very early stage of infection in the spleen and liver, but they seemed to disappear with a marked elevation of the neutralizing antibody titer. Nude mice were able to resist the virus infection when they had previously received transfer of thymocytes from weanling heterozygous littermates.     

Neurotropic viral infections leading to epilepsy: focus on Theiler's murine encephalomyelitis virus

Neurotropic viruses cause viral encephalitis and are associated with the development of seizures/epilepsy. The first infection-driven animal model for epilepsy, the Theiler's murine encephalomyelitis virus-induced seizure model is described herein. Intracerebral infection of C57BL/6 mice with Theiler's murine encephalomyelitis virus induces acute seizures from which the animals recover. However, once the virus is cleared, a significant portion of the animals that experienced acute seizures later develop epilepsy. Components of the innate immune response to viral infection, including IL-6 and complement component 3, have been implicated in the development of acute seizures. Multiple mechanisms, including neuronal cell destruction and cytokine activation, play a role in the development of acute seizures. Future studies targeting the innate immune response will lead to new therapies for seizures/epilepsy.     

Leishmania amazonensis infection in nude mice.

Leishmania amazonensis is an intracellular protozoan parasite of macrophages. Cutaneous leishmaniasis in an immunocompetent host begins as papules or nodules followed by ulceration at the site of promastigote inoculation. In this study, the pathological changes of cutaneous leishmaniasis lesions in T cell deficient nude mice were examined. When infected with L. amazonensis promastigotes, nude mice developed non-ulcerative cutaneous nodules. By histological examination of cutaneous lesions, massive accumulation of vacuolated histiocytes containing amastigotes was observed in all the nude mice. Although infiltration of mononuclear and polymorphonuclear cells was seen in the lesions of immunocompetent mice, few such cells were observed in the lesions of nude mice. These results indicate the importance of T cells on the ulcer formation in cutaneous leishmaniasis.     

Method of examining viral RNA metabolism in cells in culture: metabolism of vesicular stomatitis virus RNA.

A method is described for radioactively labeling viral RNA and then quickly halting further incorporation of radioactive precursor into RNA so that the fate of the labeled RNA can be followed. Small complementary RNAs synthesized during vesicular stomatitis virus infection in the presence of cycloheximide do not metabolize to virion length molecules when protein synthesis inhibition is reversed.     

Wild-type measles virus infection in human CD46/CD150-transgenic mice: CD11c-positive dendritic cells establish systemic viral infection

We generated transgenic (TG) mice that constitutively express human CD46 (huCD46) and/or TLR-inducible CD150 (huCD150), which serve as receptors for measles virus (MV). These mice were used to study the spreading and pathogenicity of GFP-expressing or intact laboratory-adapted Edmonston and wild-type Ichinose (IC) strains of MV. Irrespective of the route of administration, neither type of MV was pathogenic to these TG mice. However, in ex vivo, limited replication of IC was observed in the spleen lymphocytes from huCD46/huCD150 TG and huCD150 TG, but not in huCD46 TG and non-TG mice. In huCD150-positive TG mouse cells, CD11c-positive bone marrow-derived myeloid dendritic cells (mDC) participated in MV-mediated type I IFN induction. The level and induction profile of IFN-beta was higher in mDC than the profile of IFN-alpha. Wild-type IC induced markedly high levels of IFN-beta compared with Edmonston in mDC, as opposed to human dendritic cells. We then generated huCD46/huCD150 TG mice with type I IFN receptor (IFNAR1)-/- mice. MV-bearing mDCs spreading to draining lymph nodes were clearly observed in these triple mutant mice in vivo by i.p. MV injection. Infectious lymph nodes were also detected in the double TG mice into which MV-infected CD11c-positive mDCs were i.v. transferred. This finding suggests that in the double TG mouse model mDCs once infected facilitate systemic MV spreading and infection, which depend on mDC MV permissiveness determined by the level of type I IFN generated via IFNAR1. Although these results may not simply reflect human MV infection, the huCD150/huCD46 TG mice may serve as a useful model for the analysis of MV-dependent modulation of mDC response.     

Persistent vesicular stomatitis virus infection mediates base substitutions in viral RNA termini.

We have sequenced (via a product RNA) the 3' RNA terminus of a defective interfering particle that was generated from the standard virus isolated from a culture of BHK-21 cells persistently infected with vesicular stomatitis virus for over 5 years. By hybridization and RNA sequencing, seven mutations were identified in the 46 nucleotides at the terminus of this defective-interfering-particle RNA. It is likely that these mutations are a reflection of altered protein-nucleic acid interactions that the virus has evolved to maintain its persistently infected carrier state in vitro.     

Immunoglobulins and complement in demyelination induced in mice by Theiler's virus

Intracerebral inoculation of Theiler's murine encephalomyelitis virus (TMEV) produces chronic demyelination and persistent infection in the central nervous system (CNS) of susceptible SJL mice. This series of experiments examined the contribution of humoral immunity and C to myelin destruction. As in multiple sclerosis, mice persistently infected with TMEV had elevated levels of IgG and oligoclonal bands in the cerebrospinal fluid (CSF). Immunoblot studies revealed that even in animals exhibiting profound demyelination, IgG in the serum and CSF was directed primarily at virus antigen rather than at normal myelin components. Inflammatory cells positive for Ig were distributed mainly around blood vessels, but occasionally they infiltrated the spinal cord parenchyma. Rare examples of myelin sheaths positive for IgG were found by immunoelectron microscopy in spinal cord sections from infected mice; the third component of complement (C3) was commonly found in the walls of CNS blood vessels but not on myelin. Neither serum nor CSF IgG from infected mice bound to myelin sheaths or other CNS components in sections of normal syngeneic spinal cord. There were significantly more demyelinating lesions in infected mice depleted of C components with cobra venom factor. These data do not support a humoral autoimmune basis for the CNS demyelination that occurs in association with persistent TMEV infection. However, the humoral immune response directed at TMEV antigens may either limit virus spread or promote virus persistence.