Proof-of-concept study: profile of circulating microRNAs in Bovine serum harvested during acute and persistent FMDV infection

Foot-and-mouth disease virus (FMDV) is the etiological agent for foot-and-mouth disease (FMD); one of the most contagious animal diseases known. FMD targets both domestic and wild cloven-hoofed animals, and outbreaks affecting important livestock populations, such as cattle, sheep, and pigs incur enormous economic, political, and social ramifications. Traditional containment strategies combine mass vaccination with large scale animal culling. FMDV is represented globally by seven different serotypes: O, A, C, Asia1, SAT1, SAT2, and SAT3; and within each serotype there are multiple subtypes and variants. The virus is the prototypic member of the Aphthovirus genus of the family Picornaviridae and possesses a single-stranded positive-sense RNA genome of approximately 8500 nucleotides (nts), which consists of a large open reading frame (ORF) flanked by 5’ and 3’ non-translated regions (NTRs). The positive-sense RNA functions as a messenger RNA (mRNA) from which a single polyprotein is translated and subsequently proteolyzed into a series of intermediate and mature viral proteins. The ORF is traditionally sub-divided into three regions: P1, P2, and P3. The P1 region encodes four structural proteins, which assemble into the FMDV capsid, whereas the P2 and P3 regions encode the virus non-structural proteins essential for virus replication. The 5’ and 3’ NTRs flanking the ORF exhibit multiple secondary RNA structures, which regulate the stepwise translation of virus proteins and replication of the RNA genome. Knowledge obtained from the study of the molecular biology and pathogenesis of FMDV continues to be applied to the development of improved vaccines and anti-viral therapies [1, 2].

RNA interference (RNAi), a process by which mRNA transcripts are targeted for degradation or translational repression by short non-coding RNA molecules (ncRNAs) has been widely studied for its utility as an anti-viral therapeutic platform. Small interfering RNAs (siRNAs) are generated when large double-stranded RNA (dsRNA) molecules are gradually processed into progressively smaller dsRNA of approximately 20–22 nucleotides in length; and have been investigated as anti-viral therapeutics [3‐5]. In the case of FMDV, siRNAs targeted to sequences in the virus genome have been tested for their capacity to impede the progression of viral infection with reportedly mixed benefits [6‐9]. A key drawback of siRNA-based anti-viral therapies is the required complete sequence complementarity between the siRNA and its cognate RNA target. RNA viruses are highly mutable, and as such, the accumulation of mutations with each replication cycle diminishes the efficacy of the siRNA treatment. Additionally, with seven different serotypes and multiple subtypes and variants of FMDV, designing siRNAs that are universally functional across virus strains would be highly challenging.

In contrast to siRNAs, microRNAs (miRNAs) do not require perfect complementarity to their target RNA molecule [10‐13], which mitigates the effect of mutations in the RNA genome and facilitates a similar function across multiple forms of FMDV. Another key difference between siRNAs and miRNAs is that siRNAs are generally produced from exogenous RNA molecules, while miRNAs are generated from endogenous RNA molecules [12]. In the normal cellular environment, miRNAs act as key post-transcriptional regulators of gene expression for a variety of biological processes including immune response, cell proliferation, and metabolism [14‐16].

Currently, miRNAs are also being explored as potential bio-markers of infectious disease, particularly miRNAs that are detectable in the bloodstream [17‐21]. Studies have shown that different viruses generate distinct expression profiles of circulating miRNA, so much so that one group was able to distinguish between enterovirus 71 and coxsackievirus 16 as the etiological agents for individual cases of hand-foot-and-mouth disease [17]. Unfortunately, while greater than 2588 human miRNAs have been identified, only approximately 798 bovine miRNAs have been fully characterized (release 21, June 2014, miRBase.org). Furthermore, while the miRNA response to several bacterial pathogens has been examined in cattle, very few studies have been conducted in the context of viral infection [22].

Although still an evolving technology, the information gained from miRNA profiling investigations has contributed substantially to efforts for curtailing viral infection. One strategy has been to incorporate miRNA target sequences into viral genomes that target host anti-viral response pathways to contribute to the development of live attenuated vaccine platforms, which has been accomplished for vesicular stomatitis virus [23], Japanese encephalitis virus [24], coxsackievirus A21 [23], and influenza virus [25, 26]. Contrastingly, some viruses have exploited the opposite approach, as several DNA viruses and a few RNA viruses have been identified that harbor functional miRNA sequences [27‐29]. Of relevance to FMDV, miRNA sequences were identified in the anti-genome (or negative strand intermediate) of a representative Picornavirus, hepatitis A virus (HAV), thus demonstrating that RNA viruses, even those whose replication cycle is confined to the cytoplasm, can encode functional miRNAs like their DNA virus counterparts [28, 29]. Although certainly still controversial, these findings contest the notion that RNA viruses would not encode functional miRNAs since interactions with the cellular miRNA machinery would require: (i) localization of an RNA virus genome to the nucleus and (ii) that processing of the pre-miRNA hairpin would irreparably damage the virus genome.

An alternative anti-viral miRNA strategy has been to introduce artificial miRNAs in cell culture or in infected animals to target specific viral sequences or the mRNA transcripts of cellular proteins that the virus requires for an efficient replication cycle. This approach has been beneficially used in two reported instances involving FMDV, where artificial miRNAs were constructed that targeted either the IRES sequence in the 5’ NTR or the coding sequence for the 3D polymerase [30, 31]. When these constructs were transfected into cell culture prior to FMDV infection, replication was significantly impeded. In another study, the artificial miRNA sequences were inserted into the genomic DNA of a cell line, so as to allow for constitutive expression of the anti-FMDV miRNAs [32]. However, this failed to protect the cells from FMDV infection due to inefficient localization of the anti-FMDV miRNAs to sites of virus replication in the sub-cellular environment.

This current work comprises a proof-of-concept study in which the differential regulation of serum miRNAs was investigated in cattle infected with FMDV serotype A isolate A24 Cruzeiro. The bovine serum miRNA profile was characterized using quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) array plates designed to detect the relative abundance levels of 169 of the bovine miRNAs characterized in the past several years (Table 1, Fig. 1c). Serum from uninfected animals was compared to samples obtained through three distinct phases of FMDV infection: acute infection (peak viremia), persistent infection (subclinical persistence of FMDV in the upper respiratory tract), and convalescent phase, comprising animals that had successfully cleared the infection (Fig. 1a, b). Distinct miRNA signatures of up- and down-regulated miRNAs in circulation in response to FMDV infection were detected. The results obtained from this proof-of-concept study reinforce the application of this approach to future large scale testing and the investigation of miRNA signatures for other serotypes of FMDV.

In viral disease research, miRNA profiling has become a topic of considerable interest as researchers search for cellular targets that can be exploited in order to combat infection. In the case of FMDV, the sole miRNA profiling study to date was an investigation of the differential expression of miRNAs in a porcine based cell line in response to infection with an isolate of FMDV serotype Asia1 [49]. The widespread utility of these findings is somewhat limited given that cell culture is not necessarily reflective of the molecular pathogenesis of the virus in an in vivo setting. miRNA profiling analysis of tissue, urine, and serum samples from infected organisms is being conducted with greater frequency. Using miRNA RT-qPCR profiling arrays containing 169 characterized miRNAs found in Bos taurus, we investigated the differential levels of miRNAs in sera collected from cattle during three distinct phases of infection with FMDV serotype A isolate A24 Cruzeiro, including acute and persistent phases of infection. Moreover, persistent phase samples were obtained from sub-clinically infected FMDV carriers as well as convalescent animals that had successfully cleared the infection. A total of 19 miRNAs exhibited an altered abundance in serum in response to FMDV infection. Five of the miRNAs identified with significant shifts in circulating levels relative to uninfected animals were unique to acutely infected cattle (Fig. 6a, orange circle), seven were only observed in serum from persistently infected carriers (Fig. 6a, blue circle), whereas two were specific to convalescent animals (Fig. 6a, green circle). Of the miRNAs that were significantly up- or down-regulated, five were shared between acutely and persistently infected cattle (bta-miR-17-5p, bta-miR-144, bta-miR-497, bta-miR-22-5p, and bta-miR-1281). Mimics of miR-17-5p and miR-1281 were separately tested for their effect on FMDV replication in cell culture (Fig. 5). While miR-17-5p had no apparent impact on the progression of FMDV infection, mimics of miR-1281 decreased the resulting FMDV titers by 2–3 logs. From these findings, it could be inferred that the induction of miR-17-5p in vivo might manipulate the cellular environment such that it favors FMDV replication, while miR-1281 is down-regulated due to an antagonistic effect. Given that miR-1281 mimics impede FMDV replication and the reported role of miR-1281 in cattle [50], it seems that the state of lipid homeostasis in the host plays an important role in the outcome of FMDV infection regardless of whether it is acute or persistent. A survey of all the predicted target genes of these two miRNAs using multiple online algorithms failed to produce a consensus target to be explored in this current study (see Additional file 2: Table S1).

The previously reported miRNA profile of a porcine cell line infected with FMDV serotype Asia1 was obtained using high throughput sequencing [49], which represents the first time such a differential profiling experiment was performed for FMDV. The present study represents the first miRNA profiling experiment, albeit a limited small scale proof-of-concept study, using serum collected from uninfected and infected cattle. This study also marks the first time this type of analysis was performed for a serotype A FMDV isolate (A24 Cruzeiro) as well as the first time differentially expressed miRNAs were examined in the context of persistent FMDV infection. Four of the miRNAs detected were shared between the two profiling studies: miR-22-5p, miR-146b, miR-23b-5p, and miR-369-3p. Notably, these four miRNAs all participate in the regulation of cell proliferation and apoptosis. These may also potentially represent four miRNAs that are distinctive to FMDV infection across all serotypes, while the targets not shared between these two studies represent sets of miRNAs that are either specific to the FMDV strain or the experimental system (cell culture versus infected cattle). Future profiling studies with other FMDV serotypes will be necessary to bring certainty to these suppositions. We also wanted to examine the differential miRNA profiles in serum from cattle infected with other viral pathogens; however, we were unable to find published reports comparable to the proof-of-concept study described herein. Despite that, the miRNA profiles generated from human serum collected from individuals infected with related + ssRNA viruses (Dengue virus and HCV) showed some dysregulated miRNAs shared with the ones reported for FMDV here, which included: let-7 g, miR-22-5p, miR-23b-5p, miR-146a, and miR-497 [51, 52]. Of note, these shared miRNAs all are reportedly involved in the signaling pathways involved in cellular proliferation and apoptosis, from which it can be inferred that these pathways are important to life cycle of + ssRNA viruses. Potentially, these shared miRNAs that are differentially regulated in the serum in response to invasion by + ssRNA viruses suggest that common molecular mechanisms are activated or suppressed to either combat + ssRNA viruses or to alter the host environment such to facilitate + ssRNA virus replication.

There is increased evidence to support the value of the application of profiling of miRNAs detectable in circulation as biomarkers suggesting the presence and even prognosis of specific disease conditions. For example, hand-foot-and-mouth disease (HFMD) results from infection by one of many possible Picornaviruses, and research has demonstrated that each of these viruses elicits a unique systemic miRNA response that enables differentiation of the potential causative agents [17]. Thus, it would be of interest to elucidate the signature miRNA response for FMDV infection, which may be applicable across all serotypes of FMDV or could be different with each of the seven known serotypes and in different host organisms such as goats, sheep, and pigs. Similarly, identification of distinct changes in circulating miRNAs potentially associated with subclinical persistence of FMDV could be of great value for development of novel diagnostic approaches for disease surveillance. Furthermore, some of the miRNAs detected in this study might also serendipitously target regions of the FMDV genome, and could be potentially adapted as bio-therapeutic against FMDV similar to recent report with miR-203a-3p and miR-203a-5p [53]. To briefly explore that possibility, a bioinformatics analysis was conducted using two miRNA recognition sequence mapping algorithms (miRmap and ViTa) [54, 55], where the sequence for the FMDV isolate used in this study was input (A24 Cruzeiro, Accession #AY593768) along with each individual miRNA identified herein (see Additional file 3: Table S2). The miRmap algorithm reported that 6 of the 19 miRNAs dysregulated in bovine serum in response to FMDV could potentially target different regions of the FMDV A24 Cruzeiro RNA genome: bta-miR-17-5p, bta-miR-497, bta-miR-146a, bta-miR-1224, bta-miR-31, and bta-miR-150. In contrast, the ViTa algorithm found that more of these miRNAs could potentially target the genome, adding bta-miR-205, bta-miR-26b, bta-let-7 g, bta-miR-34a, bta-miR-144, bta-miR-181b, and bta-miR-147 to the list. Future studies with these new miRNA targets will certainly need to explore the relative stability of the FMDV genome in the presence of elevated levels of these select miRNAs.

While approximately 87% of the profiled miRNAs exhibited no change in circulation levels between uninfected and infected animals, roughly 13% were significantly up-regulated or down-regulated as a result of FMDV infection. Of the differentially regulated miRNAs, 16 (bta-miR-23b-5p, let-7 g, bta-miR-22-5p, bta-miR-1224, bta-miR-144, bta-miR-497, bta-miR-455-3p, bta-miR-154a, bta-miR-369-3p, bta-miR-26b, bta-miR-34a, bta-miR-205, bta-miR-181b, bta-miR-146a, bta-miR-17-5p, and bta-miR-31) have previously been described to play a role in cellular proliferation or apoptosis (Fig. 6b, orange circle). Nine of the miRNAs (bta-miR-26b, bta-miR-34a, bta-miR-205, bta-miR-181b, bta-miR-146a, bta-miR-17-5p, bta-miR-31, bta-miR-150, and bta-miR-147), have been ascribed immune modulatory functions (Fig. 6b, blue circle). Also notable is that five of the detected miRNAs (bta-miR-1281, bta-miR-369-3p, bta-miR-26b, bta-miR-34a, and bta-miR-205) are involved in adipogenesis or other lipid metabolic pathways (Fig. 6b, green circle). Interestingly, there were an equivalent number of immune modulatory miRNAs differentially expressed between acutely and persistently infected cattle with one down-regulated and three up-regulated. Only one of the immune modulatory miRNAs was shared between the two sets: bta-miR-17-5p, which was significantly upregulated during both acute and persistent FMDV infection. This miRNA has been extensively studied for its roles in immune modulation as well as cellular proliferation and apoptosis [56]. Additionally, miR-17-5p has been implicated in T-cell activation, B-cell and monocyte maturation as well as with suppression of TLR signaling and hampering of the IFN response [16, 57, 58], which are important functions of the host anti-viral response. These two test groups also exhibited approximately the same number of miRNAs associated with cell proliferation and apoptosis with eight and ten differentially expressed in acutely and persistently infected animals, respectively. Interestingly, there were two times more lipid metabolism-related miRNAs with dysregulated expression in acutely infected cattle, suggesting that modulation of such pathways is important to active FMDV replication. It is known that the VP4 structural protein is acylated with myristate [59]; and more recently, a SILAC screening showed that LYPLA, which is a known depalmitoylase, is a critical factor in FMDV infection [60]. It would be expected that the serum miRNAs of an animal that cleared FMDV would return to a basal level similar to an uninfected animal, and consistent with that, the samples obtained from convalescent cattle exhibited only two dysregulated miRNAs: one that is immune modulatory (bta-miR-150) and one that promotes cellular proliferation (bta-miR-455-3p). In serum from convalescent animals, bta-miR-150 was marginally down-regulated, possibly indicating a reduction in immune system activation given that the FMDV infection was cleared. In strong contrast, bta-miR-455-3p was the most up-regulated miRNA in all of the data sets examined in this study. A survey of published findings on miR-455-3p point toward it being a potent upstream regulator of multiple signaling pathways associated with induced cellular proliferation [61‐63]. Since one of the cellular mechanism for combating a viral infection is the induction of apoptosis in infected cells, thereby also increasing cellular turn-over, the augmentation in bta-miR-455-3p expression may be reflective of the animal reversing those effects and shutting down those pathways now that FMDV infection has been eliminated. As with the bioinformatics analysis conducted to identify prospective gene targets for miR-17-5p and miR-1281, a consensus target gene for miR-455-3p was not found to further investigate (Additional file 2: Table S1). However, consistent with the speculation above, many of the target genes proposed for miR-455-3p were cell cycle regulators and zinc-finger containing proteins, from which it can be inferred that this single miRNA was regulating multiple cell survival pathways to facilitate the final stages of clearing FMDV infection.

It is interesting to note that six of the 19 miRNAs described in this study are considerably abundant in cattle liver: bta-miR-22-5p, bta-miR-150, bta-miR-17-5p, bta-miR-455-3p, bta-miR-146, and let 7-g [64]; an organ in which FMDV does not establish infection. However, due to the substantial blood flow through the liver, large quantities of FMDV particles will pass through the liver during high titer viremia. It is unclear if or how FMDV may be affecting this tissue to possibly stimulate the secretion of the aforementioned miRNAs into the circulation. However, acute FMDV infection in cattle has previously been associated with a strong acute phase response characterized by significant induction of serum amyloid A and haptoglobin levels in serum [65]. These proteins are secreted from the liver in response to a systemic inflammatory reaction [66], which further suggests that the systemic effect of FMDV infection may affect hepatic gene expression and protein synthesis. Further investigation will be required to elucidate the extent and potential application of these findings.

In conclusion, we have identified several miRNAs that are elevated or diminished in circulation of cattle that have been infected with FMDV. Interestingly, unique miRNA serum signatures were detected for acutely infected and persistently infected cattle. The unique miRNAs found in serum from persistently infected cattle may help determine what cellular pathways are actively contributing to the development and maintenance of the “FMDV carrier state”. Characterization of signature serum miRNAs that are distinct to the FMDV carrier state could facilitate identification of persistently infected animals in post-outbreak surveillance. Moreover, the miRNAs described may also be adapted as potential anti-viral therapeutics as has been attempted with siRNAs in the past [6‐9, 67, 68]. Further investigations including increased sample numbers will be needed to verify the findings of the current proof-of-concept study. Additional in vitro investigations may provide further knowledge of distinct biological functions and potential therapeutic applications of the bovine miRNAs identified herein.

Perturbations in the circulating levels of miRNAs in the bloodstream of humans and animals in response to infection can potentially provide diagnostic and prognostic value. Here, for the first time in a small scale proof-of-concept study, differentially regulated serum miRNAs from cattle infected with FMDV at different phases of infection were reported. Signature circulating miRNAs were identified for acutely and persistently infected animals, which could be applied to the development of diagnostic assays allowing for distinction between carrier and non-carrier cattle.