Long-term persistence of West Nile virus (WNV) infection within vertebrate reservoir hosts is a potential mechanism for overwintering of this (and other) arbovirus(es) at temperate latitudes. The house sparrow (Passer domesticus), an established amplifying host for WNV and other arboviruses, was used as a model to confirm chronicity of WNV infection in passerine birds and to evaluate the feasibility of two overwintering mechanisms: blood-borne infection of arthropod vectors (recrudescence) and oral infection of vertebrate reservoir hosts (ingestion of infected tissues through predation). WNV-inoculated sparrows were monitored for persistent infection for up to 2 years. Infectious virus persisted in tissues through 43 days, but not in sera beyond 6 days. Viral RNA persisted in tissues through 65 days. Chronicity of WNV infection in some tissues, but not blood, supports the predation mechanism of WNV overwintering, but not recrudescence. RNA persistence impacts interpretation and etiologic determination of avian mortality.

Bats are reservoirs for many different coronaviruses (CoVs) as well as many other important zoonotic viruses. We sampled feces and/or anal swabs of 1,044 insectivorous bats of 2 families and 17 species from 21 different locations within Colorado from 2007 to 2009. We detected alphacoronavirus RNA in bats of 4 species: big brown bats (Eptesicus fuscus), 10% prevalence; long-legged bats (Myotis volans), 8% prevalence; little brown bats (Myotis lucifugus), 3% prevalence; and western long-eared bats (Myotis evotis), 2% prevalence. Overall, juvenile bats were twice as likely to be positive for CoV RNA as adult bats. At two of the rural sampling sites, CoV RNAs were detected in big brown and long-legged bats during the three sequential summers of this study. CoV RNA was detected in big brown bats in all five of the urban maternity roosts sampled throughout each of the periods tested. Individually tagged big brown bats that were positive for CoV RNA and later sampled again all became CoV RNA negative. Nucleotide sequences in the RdRp gene fell into 3 main clusters, all distinct from those of Old World bats. Similar nucleotide sequences were found in amplicons from gene 1b and the spike gene in both a big-brown and a long-legged bat, indicating that a CoV may be capable of infecting bats of different genera. These data suggest that ongoing evolution of CoVs in bats creates the possibility of a continued threat for emergence into hosts of other species. Alphacoronavirus RNA was detected at a high prevalence in big brown bats in roosts in close proximity to human habitations (10%) and known to have direct contact with people (19%), suggesting that significant potential opportunities exist for cross-species transmission of these viruses. Further CoV surveillance studies in bats throughout the Americas are warranted.

Viral polymerase entry and pausing at intergenic junctions is predicted to lead to a defined polarity in the levels of rhabdovirus gene expression. Interestingly, we observed that the rabies virus glycoprotein mRNA is differentially over-expressed based on this model relative to other transcripts during infection of 293T cells. During infection, the rabies virus glycoprotein mRNA also selectively interacts with the cellular poly(rC)-binding protein 2 (PCBP2), a factor known to influence mRNA stability. Reporter assays performed both in electroporated cells and in a cell-free RNA decay system indicate that the conserved portion of the 3′ UTR of the rabies virus glycoprotein mRNA contains an RNA stability element. PCBP2 specifically interacts with reporter transcripts containing this 72 base 3′ UTR sequence. Furthermore, the PCBP2 interaction is directly associated with the stability of reporter transcripts. Therefore, we conclude that PCBP2 specifically and selectively interacts with the rabies virus glycoprotein mRNA and that this interaction may contribute to the post-transcriptional regulation of glycoprotein expression.

Rabies is generally considered a fatal disease, yet neutralizing antibodies to rabies virus (RV) have frequently been found in sera from healthy, insectivorous bats, and mark-recapture studies have demonstrated bats that are still alive years after the first detection of anti-RV antibodies. To explore this phenomenon, we exposed mice to a big brown bat variant of RV, using three routes of inoculation, two doses of virus and two frequencies of exposure. We found the highest rate of seroconversion without mortality in mice that received repeated intramuscular inoculation of the higher dose of virus, and mice inoculated intranasally experienced the highest mortality.