Coxsackievirus A9 (CA9) and a variant, impaired in replication but persistently present in CSF over several months, were isolated from patients' CSF specimens during an epidemic of optic and peripheral neuropathy in Cuba in the early 1990's. The epidemic was clearly related to dietary deficiencies in several nutrients, including the antioxidants selenium, vitamin E, and lycopene. We are studying these isolates in the context of our previous work in mice, which demonstrated that deficiency of selenium or vitamin E can lead to increased severity of both Coxsackievirus B3 myocarditis and influenza pneumonia, with concomitant mutations in the viral genome. These mutations are reproducible, and the mutant strains have increased virulence for mice with normal nutriture. We hypothesize that host nutritional deficiencies induced mutations in CA9, leading to a new viral variant with altered pathogenic potential. We have completed genomic sequence analysis of three Cuban CA9 isolates from neuropathy patients and of five meningitis isolates from before the epidemic. Phylogenetic analysis shows the neuropathy isolates to be different, with a unique mutation near the active site of 2a protease that may contribute to the altered capsid proteins previously demonstrated in the variant virus. Using homologous regions among the CA9 already sequenced, we designed improved primers for sequencing the variant, which required special methods for RNA isolation because of apparent instability and/or very limited quantity. We have produced full-length cDNA from the variant, and sequences in the 5' non-translated region are consistent with a CA9 not identical to the other isolates. Using electron microscopy, we have demonstrated typical picornavirus virions in thin sections of CA9, but not of the variant, although the latter produces abundant viral antigen as demonstrated by immunofluorescence with homologous antiserum. We have produced mutations in CB3 by in vitro exposure to hydrogen peroxide in cell culture, and we will now apply the methods developed to CA9. In this continuation proposal, our objectives are 1) to characterize the quasispecies populations of the CA9 and variant isolates from Cuban patients; 2) to determine the role of the 2a protease and other mutations in altering the capsid proteins and replication patterns of the variant; 3) to determine the effects of repeated passage of CA9 and variant viruses in cells exposed to, or protected from, oxidative stress; and 4) to study the capsid morphology of the variant by comparing it to normal CA9 using cryo-electron microscopy. ? ?

National Institute of Health (NIH)
National Eye Institute (NEI)
Research Project (R01)
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Nutrition Study Section (NTN)
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Hunter, Chyren
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University of North Carolina Chapel Hill
Schools of Medicine
Chapel Hill
United States
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