•CVB3 RNA persists in mouse hearts for weeks after clearance of infectious virus.•Although abundant, this RNA triggers neither T1IFN responses nor local inflammation.•After RNA persistence was established, T1IFNR was ablated in vivo from cardiomyocytes.•This had no effect on RNA levels, and infectious virus did not emerge.Type B coxsackieviruses (CVB) can cause myocarditis and dilated cardiomyopathy (DCM), a potentially-fatal sequela that has been correlated to the persistence of viral RNA. Herein, we demonstrate that cardiac RNA persistence can be established even after an inapparent primary infection. Using an inducible Cre/lox mouse model, we ask: (i) Does persistent CVB3 RNA cause ongoing immune activation? (ii) If T1IFN signaling into cardiomyocytes is ablated after RNA persistence is established, is there any change in the abundance of persistent CVB3 RNA and/or does cytopathic infectious virus re-emerge? (iii) Does this loss of T1IFN responsiveness by cardiomyocytes lead to the recurrence/exacerbation of myocarditis? Our findings suggest that persistent enteroviral RNAs probably do not contribute to ongoing myocardial disease, and are more likely to be the fading remnants of a recent, possibly sub-clinical, primary infection which may have set in motion the process that ultimately ends in DCM.

Transgenic mice expressing a tamoxifen-inducible Cre recombinase specifically in cardiomyocytes were generated in 2001 and are in widespread use, having been employed in >150 published studies. However, several groups recently have reported that tamoxifen administration to these mice can have off-target effects that include cardiac dysfunction, fibrosis, and death. For this reason, among others, we considered it important to better characterize the transgene (termed herein, CM-MCM) and its chromosomal location(s). Cytogenetic analysis positioned the CM-MCM transgene within the C band of chromosome 19, and more precise mapping, using genome walking and DNA sequencing, showed that transgene insertion is in the C1 region. Using the genome walking data, we have developed PCR assays that not only identify mice that carry the transgene, but also distinguish homozygous animals (CM-MCMTg/Tg) from hemizygous (CM-MCMTg/0), permitting the rapid assessment of transgene zygosity and, thereby, helping to minimize off-target tamoxifen-induced effects. Substantial rearrangement/duplication of transgene elements is present, and transgene integration was accompanied by the deletion of a 19,500 bp fragment of genomic DNA that contains the promoter, exon 1 and part of intron 1 of the APOBEC1 complementation factor (A1cf) gene, as well as several elements that are predicted to regulate chromosomal architecture. A1cf protein expression is ablated by the deletion and, therefore, homozygous mice are functionally A1cf knockout. The implications of this unexpected finding are discussed.

The family Picornaviridae contains some notable members, including rhinovirus, which infects humans more frequently than any other virus; poliovirus, which has paralysed or killed millions over the years; and foot-and-mouth-disease virus, which led to the creation of dedicated institutes throughout the world. Despite their profound impact on human and animal health, the factors that regulate pathogenesis and tissue tropism are poorly understood. In this article, we review the clinical and economic challenges that these agents pose, summarize current knowledge of host–pathogen interactions and highlight a few of the many outstanding questions that remain to be answered.

CD8+ memory T cells produce IFNγ within hours of secondary infection, but this is quickly terminated in vivo despite the presence of stimulatory viral antigen, suggesting that active suppression occurs. Herein, we investigated the in vivo effector function of CD8+ memory T cells during successive encounters with viral antigen. CD8+ T cells in immune mice receiving prior viral or peptide challenge failed to reproduce IFNγ during LCMV rechallenge. Surprisingly, this refractory state was induced even in memory cells that had not encountered their cognate antigen, indicating that the silencing of CD8+ T cell responses is TCR-independent. Direct injection of IFNγ also suppressed the ability of virus-specific memory cells to respond to subsequent viral challenge. We propose the existence of a negative feedback loop whereby IFNγ, produced by memory CD8+ T cells to combat viral challenge, acts – directly or indirectly – to limit its further production.

Key features of an ideal RNA-based vaccine against coxsackievirus B3 (CVB3) are (i) limited genome replication/virus production (to minimize vaccine-related pathology) and (ii) abundant virus protein synthesis (to maximize immunogenicity). These attributes may apply to CVB3 RNAs lacking up to 250 nucleotides (nt) from their 5′ terminus; these RNAs do not give rise to infectious progeny, but they have been reported to retain the entire CVB3 IRES (mapped to nt ∼ 432–639) and to produce large quantities of viral protein in transfected cells. Here, we constructed five 5′ RNA deletion variants that, to our surprise, failed to protect against CVB3 challenge. We investigated the reasons for this failure and conclude that (i) a 5′ terminal deletion as short as 32 nt abolishes CVB3 RNA replication in transfected cells; (ii) this deleted RNA, and others with longer deletions, do not direct abundant protein synthesis in transfected cells, probably as a consequence of their replicative incapacity; and (iii) the CVB3 IRES is substantially larger than previously thought, and its 5′ boundary lies between residues 76 and 125, very closely approximating that of the poliovirus IRES.