Alphacoronaviruses in New World Bats: Prevalence, Persistence, Phylogeny, and Potential for Interaction with Humans

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

The 3′ Untranslated Region of the Rabies Virus Glycoprotein mRNA Specifically Interacts with Cellular PCBP2 Protein and Promotes Transcript Stability

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

Characterization of big brown bat (Eptesicus fuscus) rabies virus in a mouse model

2011 Fall.Includes bibliographical references.A majority of human rabies cases in the United States are either imported from countries where dog rabies is endemic or classified as cryptic human cases, where a route of exposure is not known. Notably, essentially all rabies virus (RV) variants associated with cryptic cases of human rabies are maintained in bats. Understanding how RV is maintained in populations of bats and characterizing the diversity of bat RV is thus a high priority problem for public health. Among the knowledge gaps related to bat rabies are understanding the variation in virulence within the population of a single variant and explaining the observation that a substantial number of healthy wild bats have neutralizing antibodies to RV, but no apparent clinical illness. The work described here was designed to address both of those issues. Nine RV isolates were isolated from big brown bats in Colorado and low-passage stocks of each were prepared. These isolates were evaluated for virulence, immunogenicity and salivary gland dissemination to investigate whether there were major differences in these characteristics within this virus population. Inoculated mice were maintained for 12 weeks after virus inoculation to assess mortality and were bled regularly to evaluate their humoral immune responses. Salivary glands from mice that developed clinical rabies were evaluated for virus replication as an indication for potential for further transmission. The dose of RV inoculated had a greater influence on the incubation period and mortality than the individual RV isolate. There was no difference in the humoral immune response in mice between those that were protected and those that succumbed to infection. The only salivary glands that were positive for RV replication were observed from mice in the high dose inoculation groups. Collectively, the results of this experiment indicated that there was low diversity in biologic behavior within the sample of Epitesicus fuscus viruses tested. The humoral immune response of mice to a big brown bat RV variant was explored to address the hypothesis that dose, route or frequency of inoculation may explain the prevalence of neutralizing rabies antibody seen in wild bat populations. Mice were inoculated via intramuscular, intradermal and intranasal routes, with two different low doses of virus and two inoculation schedules. The highest frequency of seroconversion was seen in mice inoculated intramuscularly with the higher of the two doses of RV. Mice that were inoculated intranasally experienced the highest mortality. Mice were rechallenged 3 months following the initial challenge with a high dose of virus intramuscularly to determine if the neutralizing rabies antibodies were protective and if priming of the immune system to RV had occurred in those that failed to seroconvert. The results of this experiment indicate that inoculation of low doses of virus by any of several routes can elicit a detectable humoral immune response without development of disease, which supports the hypothesis that exposure of wild bats to low doses of RV results in seroconversion without clinical disease

The 3′ UTR of the rabies virus G mRNA represses deadenylation of a reporter RNA in a cell free deadenylation/decay assay.

<p>Panel A: Radiolabeled capped and polyadenylated RNAs containing either reporter only (Gem A60) or the reporter plus the 3′ UTR of the rabies virus P, (P 3′ UTR), M, (M 3′ UTR) or G (G 3′ UTR) mRNAs were incubated with HeLa cytoplasmic extract in a cell-free deadenylation/decay assay. Aliquots were taken at the times indicated and reaction products were analyzed on a 5% polyacrylamide gel containing urea. The position of the polyadenylated (A₆₀) input RNA is indicated in the ‘0’ lane and a deadenylated marker RNA is run in the ‘A₀’ lane. Panel B: Graphical representation of three independent experiments as described in Panel A. The error bars represent standard deviations.</p

The protein responsible for stabilizing reporter RNAs containing the 3′ UTR of the rabies virus G mRNA is a poly(C) binding protein.

<p>Panel A. Radiolabeled, capped and polyadenylated reporter RNA containing the 3′ UTR of the rabies virus G mRNA was incubated in the cell-free RNA deadenylation/decay system using HeLa cytoplasmic extract for 10 minutes in the presence of the indicated amount of poly(C) competitor RNA. Reaction products were analyzed on a 5% polyacrylamide gel containing urea. The positions of the input polyadenylated (A₆₀) and deadenylated product (A₀) are indicated at the left of the gel. The lane marked input indicates the RNA in a reaction that was set up but not incubated. Panel B: Same as panel A. 2.5 µgs of the indicated competitor RNA were added in the lanes marked poly C and poly G. The lane marked ‘0’ indicates a control deadenylation reaction to which no competitor RNA was added. Reactions were incubated for 10 minutes. Panel C: HeLa cytoplasmic extracts were immunodepleted using the indicated antisera. In the left panel, the extent of immunodepletion was examined by western blot using the indicated antisera. GAPDH was used as a normalization control. In the right panel, extracts immunodepleted using either control IgG or PCBP2-specific antibodies were used in our cell-free deadenylation assays with a reporter RNA containing the 3′ UTR of the G mRNA. Samples were taken at the times indicated and analyzed on a 5% polyacrylamide gel containing urea. The positions of the input polyadenylated (A₆₀) and deadenylated product (A₀) are indicated at the left of the RNA gel.</p

The cellular PCBP2 protein selectively interacts with the G mRNA during rabies virus infection.

<p>Rabies virus infected 293T cells were treated with formaldehyde to stabilize RNA-protein complexes and cell lysates were immunoprecipitated using either control IgG or a PCBP2-specific antisera. Co-precipitating rabies viral mRNAs were analyzed by RT-PCR after reversal of the formaldehyde cross links and products run on a 1% agarose gel and visualized with ethidium bromide. The 10% input lane represents total RNA present in the lysate prior to immunoprecipitation. The numbers below the lanes represent quantification and standard deviation from three independent experiments.</p

Electroporated reporter RNAs can be stabilized by a conserved segment of the rabies virus G 3′UTR.

<p>A radiolabeled, capped and polyadenylated reporter RNA derived from pGem-A60 (reporter only), or a variant containing a 72 base segment from the 3′ UTR of the rabies virus G mRNA (reporter+G 3′ UTR), were electroporated into BHK cells. Total RNA was isolated from cells at the times indicated, analyzed on a 5% polyacrylamide gel containing urea, and visualized by phosphorimaging. A graphical representation of the results obtained independent representative experiment is shown. The standard deviation shown in the calculation of half-life differences was determined from 3 independent experiments.</p