Coxsackievirus can persist in murine pancreas by deletion of 5' terminal genomic sequences.

Enterovirus infections are generally acute and rapidly cleared by the host immune response. Enteroviruses can at times persist in immunologically intact individuals after the rise of the type-specific neutralizing immune response. The mechanism of enterovirus persistence was shown in group B coxsackieviruses (CVB) to be due to naturally-occurring deletions at the 5' terminus of the genome which variably impact the stem-loop secondary structure called domain I. These deletions result in much slower viral replication and a loss of measurable cytopathic effect when such 5' terminally deleted (TD) viruses are assayed in cell culture. The existence and persistence of CVB-TD long after the acute phase of infection has been documented in hearts of experimentally inoculated mice and naturally infected humans but to date, the existence of TD enteroviral populations have not been documented in any other organ. Enteroviral infections have been shown to impact type 1 diabetes (T1D) onset in humans as well as in the non-obese diabetic mouse model of T1D. The first step to studying the potential impact of CVB-TD on T1D etiology is to determine whether CVB-TD populations can arise in the pancreas. After inoculation of NOD diabetic mice with CVB, viral RNA persists in the absence of cytopathic virus in pancreas weeks past the acute infectious period. Analysis of viral genomic 5' termini by RT-PCR showed CVB-TD populations displace the parental population during persistent replication in murine pancreata.

Development of a new oral poliovirus vaccine for the eradication end game using codon deoptimization.

Enormous progress has been made in global efforts to eradicate poliovirus, using live-attenuated Sabin oral poliovirus vaccine (OPV). However, as the incidence of disease due to wild poliovirus has declined, vaccine-derived poliovirus (VDPV) has emerged in areas of low-vaccine coverage. Coordinated global cessation of routine, type 2 Sabin OPV (OPV2) use has not resulted in fewer VDPV outbreaks, and continued OPV use in outbreak-response campaigns has seeded new emergences in low-coverage areas. The limitations of existing vaccines and current eradication challenges warranted development of more genetically stable OPV strains, most urgently for OPV2. Here, we report using codon deoptimization to further attenuate Sabin OPV2 by changing preferred codons across the capsid to non-preferred, synonymous codons. Additional modifications to the 5' untranslated region stabilized known virulence determinants. Testing of this codon-deoptimized new OPV2 candidate (nOPV2-CD) in cell and animal models demonstrated that nOPV2-CD is highly attenuated, grows sufficiently for vaccine manufacture, is antigenically indistinguishable from Sabin OPV2, induces neutralizing antibodies as effectively as Sabin OPV2, and unlike Sabin OPV2 is genetically stable and maintains an attenuation phenotype. In-human clinical trials of nOPV2-CD are ongoing, with potential for nOPV strains to serve as critical vaccine tools for achieving and maintaining polio eradication.

Reversion to wildtype of a mutated and nonfunctional coxsackievirus B3CRE(2C).

The cis-acting replication element (CRE) in the 2C protein coding region [CRE(2C)] of enteroviruses (EV) facilitates the addition of two uridine residues (uridylylation) onto the virus-encoded protein VPg in order for it to serve as the RNA replication primer. We demonstrated that coxsackievirus B3 (CVB3) is replication competent in the absence of a native (uridylylating) CRE(2C) and also demonstrated that lack of a functional CRE(2C) led to generation of 5' terminal genomic deletions in the CVB3 CRE-knock-out (CVB3-CKO) population. We asked whether reversion of the mutated CRE(2C) occurred, thus permitting sustained replication, and when were 5' terminal deletions generated during replication. Virions were isolated from HeLa cells previously electroporated with infectious CVB3-CKO T7 transcribed RNA or from hearts and spleens of mice after transfection with CVB3-CKO RNA. Viral RNA was isolated in order to amplify the CRE(2C) coding region and the genomic 5' terminal sequences. Sequence analysis revealed reversion of the CVB3-CKO sequence to wildtype occurs by 8 days post-electroporation of HeLa cells and by 20days post-transfection in mice. However, 5' terminal deletions evolve prior to these times. Reversion of the CRE(2C) mutations to wildtype despite loss of the genomic 5' termini is consistent with the hypothesis that an intact CRE(2C) is inherently vital to EV replication even when it is not enabling efficient positive strand initiation.

A novel, broadly applicable approach to isolation of fungi in diverse growth media.

Creatinine (CRN) is a vertebrate metabolic waste product normally found in blood and urine. Previous work demonstrated that the hydrochloride salt of creatinine (CRN-HCl) acted as a potent inhibitor of bacterial replication. Creatinine hydrochloride does not inhibit the growth of yeasts or molds (i.e. fungi), making it a potentially useful addition to growth media to facilitate isolation of environmental or clinically relevant fungal species. Sabouraud dextrose agar is the current medium of choice for detection and isolation of fungi although it does not offer optimal nutritional requirements for some fungi and can permit growth of bacteria which may subsequently inhibit fungal growth and/or obscure fungal isolation. We show that CRN-HCl effectively suppresses bacterial growth in either liquid or solid agar media while allowing outgrowth of slower growing fungi using either experimentally prepared samples or environmental samples.