Central nervous system demyelination and remyelination in multiple sclerosis and viral models of disease

The mechanisms of myelin injury and repair were studied in acute multiple sclerosis lesions and in a murine model of demyelination induced by a virus. Injury to oligodendrocytes resulting in degeneration of inner glial loops and inner myelin lamellae (dying-back oligodendrogliopathy) was observed by electron microscopy in brain biopsies of acute demyelinating lesions. Attempts at central nervous system remyelination as manifested by thinly myelinated axons and proliferation of oligodendrocytes were observed at the edge of many acute plaques. To develop therapeutic strategies to inhibit demyelination or promote remyelination, mice infected intracranially with Theiler's virus (a picornavirus) were studied. Experimental manipulation of Theiler's virus-infected mice by treatment during chronic demyelinating disease with immunoglobulins directed at normal spinal cord antigens or with monoclonal antibodies which deplete CD4 or CD8-positive T cells reslted in augmentation of new myelin synthesis. These observations suggest that disturbances in the myelinating function of oligodendrocytes, events not accompanied by death of these cells, may be among the earliest pathological events in multiple sclerosis. Experiments using the Theiler's virus model of demyelination indicate that manipulation of the immune response has the potential to promote central nervous system remyelination and functional recovery in multiple sclerosis.     

Molecular epidemiology of enteroviruses causing uveitis and multisystem hemorrhagic disease of infants

We studied molecular epidemiology of highly virulent echovirus 11 and 19 strains that were isolated during five outbreaks of enterovirus uveitis (EU) in Siberia in 1980-1989, and three outbreaks of multisystem hemorrhagic disease of infants (MHD) in 1988-1991. Three genome regions, 5'NTR, VP1-2A junction, and a fragment of 3D polymerase, were analyzed. Phylogenetic grouping in the VP1-2A region correlated with serotyping results. All studied EV11 and EV19 strains, including the prototype EV11 and EV19, formed a major phylogenetic group in VP1-2A region. Within that group, several EV11 isolates from EU and MHD outbreaks formed a distinct cluster in VP1-2A and 5' NTR genome regions, designated EV11/B. All strains of this cluster possessed high virulence for monkeys compared with the prototype echoviruses. Subgrouping within this cluster correlated with year of virus isolation, not with the disease the viruses caused in infants (EU or MHD).     

Neutralization epitope diversity of coxsackievirus B4 isolates detected by monoclonal antibodies

Only recently have we begun to fully realize the diversity of virions within a single human isolate of the group B Coxsackieviruses. These intratypic (strain) differences may be one of the important factors influencing pathogenesis, e.g., myocarditis vs. pancreatitis. Yet, until the virion strains within a type can be well differentiated, a thorough analysis of the pathogenic potential of each is not possible. We compared two human isolates, JVB and Edwards, and 15 isolates derived from these two in search of determinants of strain specificity and to evaluate the diversity/stability of neutralization epitopes on the virions comprising each isolate. Polyclonal antisera failed to show strain specific determinants. However, monoclonal antibodies directed to individual neutralization epitopes defined strain specific differences. Plaque reduction neutralization tests with these monoclonal antibodies allowed the quantitation of the expression of epitopes on the virions in each isolate. These data helped to establish the limits of diversity and stability of the neutralization epitopes of Coxsackievirus B4 virions and describe the changes in epitopes among laboratory isolates.     

Degradation of the encephalomyocarditis virus and hepatitis A virus 3C proteases by the ubiquitin/26S proteasome system in vivo

We have isolated stably transfected mouse embryonic fibroblast cell lines that inducibly express either the mature encephalomyocarditis virus (EMCV) or hepatitis A virus (HAV) 3C protease and have used these cells to demonstrate that both proteins are subject to degradation in vivo by the ubiquitin/26S proteasome system. The detection of 3C protease expression in these cells requires inducing conditions and the presence of one of several proteasome inhibitors. Both 3C proteases are incorporated into conjugates with ubiquitin in vivo. HAV 3C protease expression has deleterious effects on cell viability, as determined by observation and counting of cells cultured in the absence or presence of inducing conditions. The EMCV 3C protease was found to be preferentially localized to the nucleus of induced cells, while the HAV 3C protease remains in the cytoplasm. The absence of polyubiquitinated EMCV 3C protease conjugates in nuclear fraction preparations suggests that localization to the nucleus can protect this protein from ubiquitination.     

Enterovirus A71 antivirals: Past,present, and future

Enterovirus A71 (EV-A71) is a significant human pathogen, especially in children. EV-A71 infection is one of the leading causes of hand, foot, and mouth diseases (HFMD), and can lead to neurological complications such as acute flaccid myelitis (AFM) in severe cases. Although three EV-A71 vaccines are available in China, they are not broadly protective and have reduced efficacy against emerging strains. There is currently no approved antiviral for EV-A71. Significant progress has been made in developing antivirals against EV-A71 by targeting both viral proteins and host factors. However, viral capsid inhibitors and protease inhibitors failed in clinical trials of human rhinovirus infection due to limited efficacy or side effects. This review discusses major discoveries in EV-A71 antiviral development, analyzes the advantages and limitations of each drug target, and highlights the knowledge gaps that need to be addressed to advance the field forward.     

Genome characterization of a novel chicken picornavirus distantly related to the members of genus Avihepatovirus with a single 2A protein and a megrivirus-like 3′ UTR

The members of the genus Avihepatovirus and related picornaviruses (“Aalivius”) of ducks, turkey and chickens possess identical 2A peptide composition including three functionally unrelated 2A peptides which is a characteristic genome feature of these monophyletic avian picornaviruses. The complete genome of a novel picornavirus provisionally named Orivirus A1 (KM203656) from a cloacal sample of a 4-week-old diarrheic chicken (Gallus gallus domesticus) distantly related to members of genus Avihepatovirus was characterized. The study strain contains a type-II-like IRES, a single 2A protein of unknown function unrelated to the 2A proteins of avihepatoviruses and a long 3′ untranslated region (UTR) with multiple repeated sequence motifs followed by an AUG-rich region. The repeated sequences of the 3′ UTR show significant identity to the “Unit A” sequences of the phylogenetically distant megriviruses. The presence of a novel single 2A and the megrivirus-like “Unit A” motifs suggest multiple recombination events in the evolution of this novel picornavirus.     

Amino acid substitutions within the 2C coding sequence of Theiler's Murine Encephalomyelitis virus alter virus growth and affect protein distribution

Theiler's murine encephalomyelitis virus (TMEV) was used to investigate the distribution of P2 proteins in host cells and examine the effect of amino acid substitutions in conserved residues of the 2C protein on virus growth. The distribution of viral proteins 2B, 2C and 2BC with marker proteins of the endoplasmic reticulum (ER) and/or Golgi suggest an association with membranes of the secretory pathway. Similar results were obtained for truncated 2C and 2BC proteins with C-terminal deletions suggesting that the N-terminal region of the 2C protein is important in dictating distribution patterns. The significance of the high degree of conservation of this 2C region throughout the Picornaviridae was investigated by substituting conserved amino acid residues for alanine to create six mutant strains. Substitution mutations E(8)A, W(18)A and W(29)A abolished the ability of the virus to induce cytopathic effect (CPE) in BHK-21 cells. K(14)A, R(4)A and I(23)A delayed the onset and progression of CPE compared to the wild-type (WT) virus, and decreased virus yield. Immunofluorescence analysis of cells transiently expressing mutant 2C proteins revealed that the distribution of 2C was affected by substituting K(14), W(18) and I(23) for alanine indicating that specific conserved residues in 2C dictate protein distribution and virus growth.     

The structure and function of a cis-acting element located upstream of the IRES that influences Coxsackievirus B3 RNA translation

We have investigated the importance of a conserved hexa-nucleotide stretch in the apical loop within stem-loop C (SL C, nt 104-180), upstream of the ribosome landing site, on CVB3 IRES function. The deletion or substitution mutation at this apical loop resulted in significant decrease in IRES activity. Both the mutant IRES RNAs failed to interact with certain trans-acting factors. Furthermore, expression of CVB3 2A protease significantly enhanced IRES activity of the wild type, but the effect was not so pronounced on the mutant IRESs. It is possible that the mutant RNAs were unable to interact with some trans-acting factors critical for enhanced IRES function. Interestingly, the local structure of the IRES RNA was not significantly altered due to substitution mutation. Taken together, it appears that the SL C/c apical loop is critical for CVB3 IRES function.     

Pathogenesis of virus-induced immune-mediated demyelination

Theiler's murine encephalomyelitis virus-induced demyelinating disease has been extensively studied as an attractive infectiousmodel for human multiple sclerosis. Virus-specific inflammatory Th1 cell responses followed by autoimmune responses to myelin antigens play a crucial role in the pathogenic processes leading to demyelination. Antibody and cytotoxic T cells (CTL) responses to virus appears to be primarily protective from demyelinating disease. Although the role of Th1 and CTL responses in the induction of demyelinating disease is controversial, assessment of cytokine sproduced locally in the central nervous system (CNS) during the course of disease and the effects of altered inflammatory cytokine levels strongly support the importance of Th1 responses in this virus-induced demyelinating disease. Induction of various chemokines and cytokines in different glial and antigen presenting cells upon viral infection appears to be an important initiation mechanism for inflammatory Th1 responses in the CNS. Coupled with the initial inflammatory responses, viral persistence in the CNS may be acritical factor for sustaining inflammatory responses and consequent immune-mediated demyelinating disease.